Novel and efficient method for culturing patient-derived gastric cancer stem cells.

Experimental techniques for patient-derived cancer stem-cell organoids/spheroids can be powerful diagnostic tools for personalized chemotherapy. However, establishing their cultures from gastric cancer remains challenging due to low culture efficiency and cumbersome methods. To propagate gastric cancer cells as highly proliferative stem-cell spheroids in vitro, we initially used a similar method to that for colorectal cancer stem cells, which, unfortunately, resulted in a low success rate (25%, 18 of 71 cases). We scrutinized the protocol and found that the unsuccessful cases were largely caused by the paucity of cancer stem cells in the sampled tissues as well as insufficient culture media. To overcome these obstacles, we extensively revised our sample collection protocol and culture conditions. We then investigated the following second cohort and, consequently, achieved a significantly higher success rate (88%, 29 of 33 cases). One of the key improvements included new sampling procedures for tumor tissues from wider and deeper areas of gastric cancer specimens, which allowed securing cancer stem cells more reproducibly. Additionally, we embedded tumor epithelial pieces separately in both Matrigel and collagen type-I as their preference to the extracellular matrix was different depending on the tumors. We also added a low concentration of Wnt ligands to the culture, which helped the growth of occasional Wnt-responsive gastric cancer stem-cell spheroids without allowing proliferation of the normal gastric epithelial stem cells. This newly improved spheroid culture method may facilitate further studies, including personalized drug-sensitivity tests prior to drug therapy.

Lineage tracing and targeting of IL17RB+ tuft cell-like human colorectal cancer stem cells.

Cancer stem cell (CSC)-specific markers may be potential therapeutic targets. We previously identified that Dclk1, a tuft cell marker, marks tumor stem cells (TSCs) in mouse intestinal adenomas. Based on the analysis of mouse Dclk1+ tumor cells, we aimed to identify a CSC-specific cell surface marker in human colorectal cancers (hCRCs) and validate the therapeutic effect of targeting it. IL17RB was distinctively expressed by Dclk1+ mouse intestinal tumor cells. Using Il17rb-CreERT2-IRES-EGFP mice, we show that IL17RB marked intestinal TSCs in an IL13-dependent manner. Tuft cell-like cancer cells were detected in a subset of hCRCs. In these hCRCs, lineage-tracing experiments in CRISPR-Cas9-mediated IL17RB-CreERT2 knockin organoids and xenograft tumors revealed that IL17RB marks CSCs that expand independently of IL-13. We observed up-regulation of POU2F3, a master regulator of tuft cell differentiation, and autonomous tuft cell-like cancer cell differentiation in the hCRCs. Furthermore, long-term ablation of IL17RB-expressing CSCs strongly suppressed the tumor growth in vivo. These findings reveal insights into a CSC-specific marker IL17RB in a subset of hCRCs, and preclinically validate IL17RB+ CSCs as a cancer therapeutic target.

Lower Incidence of Postoperative Pulmonary Complications Following Robot-Assisted Minimally Invasive Esophagectomy for Esophageal Cancer: Propensity Score-Matched Comparison to Conventional Minimally Invasive Esophagectomy.

BACKGROUND: Whether robot-assisted minimally invasive surgery (RAMIE) is more beneficial than conventional minimally invasive surgery (MIE) remains unclear. METHODS: In total, 165 consecutive patients with esophageal carcinoma who underwent esophagectomy between January 2015 and April 2020 were retrospectively assessed. A 1:1 propensity score matching analysis was performed to compare the short-term outcomes between RAMIE and conventional MIE. RESULTS: After matching, 45 patients were included in the RAMIE and conventional MIE groups. RAMIE had a significantly longer total operative time (708 vs. 612 min, P < 0.001) and thoracic operative time (348 vs. 285 min, P < 0.001) than conventional MIE. However, there were no significant differences in terms of oncological outcomes, such as R0 resection rate and number of resected lymph nodes. The overall postoperative morbidity (Clavien-Dindo [C-D] grade II or higher) rate of RAMIE and conventional MIE were 51% and 73% (P = 0.03), respectively, and the severe postoperative morbidity (C-D grade III or higher) rates were 11% and 29% (P = 0.04), respectively. The incidence rate of recurrent laryngeal nerve palsy was halved in RAMIE (7%) compared with conventional MIE (20%) (P = 0.06). Finally, the pulmonary complication rate (18%) was significantly lower in patients who underwent RAMIE than in those who underwent conventional MIE (44%) (P = 0.006). CONCLUSIONS: RAMIE was safe and feasible, even during the early period of its application at a specialized center. Moreover, it may be a promising alternative to conventional MIE, with better short-term outcomes, including significantly lower incidence of pulmonary complications.

Long-Term Outcomes of Laparoscopic Radical Gastrectomy for Highly Advanced Gastric Cancer: Final Report of a Prospective Phase II Trial (KUGC04).

BACKGROUND: This is the final report evaluating the long-term outcomes of a single-arm phase II clinical trial that demonstrated the short-term efficacy of laparoscopic gastrectomy (LG) for highly advanced gastric cancer (AGC) [KUGC04]. PATIENTS AND METHODS: Seventy-three patients with histologically confirmed gastric adenocarcinoma and diagnosed with clinical stage II or higher, who potentially underwent curative resection between August 2009 and November 2014, were prospectively enrolled. Long-term outcomes with 5-year progression-free survival (PFS) and 5-year overall survival (OS) were evaluated according to clinical or pathological stages. Recurrence and progression patterns were also investigated. These outcomes were compared with those of previous reports to assess the applicability of LG for highly advanced gastric cancer (HAGC). RESULTS: The median observation period of all surviving patients was 75.1 months. The 5-year PFS and 5-year OS of all patients was 47.4% and 54.4%, respectively. Clinical stage-specific 5-year PFS and 5-year OS was 75.0, 69.1, 53.9, 39.4, 40.0 and 9.1, and 75.0, 68.8, 61.5, 45.0, 60.0 and 27.3, respectively, in stages IIA, IIB, IIIA, IIIB, IIIC, and IV, respectively. Pathological stage-specific 5-year PFS and 5-year OS, including ypStage with preoperative chemotherapy, was 100, 80.0, 100, 62.5, 80.0, 51.3, 16.7, 22.2 and 12.5, and 100, 80.0, 100, 75.0, 80.0, 64.2, 25.0, 33.3 and 12.5, respectively, in stage X (no residual tumor with preoperative chemotherapy), IA, IB, IIA, IIB, IIIA, IIIB, IIIC, and IV, respectively. Recurrence or progression was observed in 30 patients (41.1%). CONCLUSION: LG for HAGC performed by experienced surgeons is safe and oncologically acceptable.

Effect of antiplatelet therapy on patients undergoing gastroenterological surgery: thromboembolic risks versus bleeding risks during its perioperative withdrawal.

BACKGROUND: Antiplatelet agents given to prevent thromboembolic disease are frequently withdrawn prior to surgical procedures to reduce bleeding complications. This action may expose patients to increased thromboembolic morbidity and mortality. METHODS: A series of 2012 patients who had undergone gastroenterologic surgery between January 2005 and June 2010 at our institution were reviewed. Among this cohort, antiplatelet therapy (APT) was used in 519 patients (25.8 %). The perioperative management included interruption of APT 1 week before surgery and early postoperative reinstitution in patients at low thromboembolic risk, although APT was maintained until surgery in those at high thromboembolic risk. Bleeding and thromboembolic complications, as well as other outcome variables, were assessed in patients with and without APT. RESULTS: Among 519 patients with APT, 99 (19.1 %) underwent multidrug APT. Among them, 124 (23.9 %) required preoperative continuation of APT. None suffered from excessive bleeding intraoperatively. There were 19 thromboembolic events (0.9 %) in the whole cohort. Postoperative bleeding complications occurred in 37 patients (1.8 %). Multivariate analysis showed that increased postoperative bleeding complications were independently associated with multidrug APT [hazard ratio (HR) 4.3, p = 0.014], high-risk surgical procedures (HR 3.5, p = 0.003), and perioperative heparin bridging (HR 2.8, p = 0.029). High-risk surgery (HR 8.3, p < 0.001) and poor performance status (HR 4.9, p = 0.005)--but neither APT nor anticoagulation use--were significant prognostic factors for thromboembolic complications. CONCLUSIONS: Satisfactory outcomes were obtained during gastroenterologic surgery under rigorous perioperative management, including single-agent APT continuation in patients at high thromboembolic risk. Patients treated with multidrug APT still represent a challenging group, however, and need to be carefully managed to prevent perioperative complications.

[A case of adenocarcinoma occurring in the bladder mucosa after a surgical operation for colovesical fistula].

We report a case of adenocarcinoma occurring in the bladder mucosa 6 years after a surgical operation for colovesical fistula due to colonic diverticulitis of the sigmoid colon. The patient was a 76-year-old woman who had undergone a sigmoidectomy and ligation of the colovesical fistula at the age of 70 years. She presented with a complaint of gross hematuria. Cystoscopy and computed tomography revealed bladder cancer at the site of the original colovesical fistula surgery. She underwent transurethral resection of the bladder tumor. Histopathological findings revealed intestinal adenocarcinoma in the urinary bladder. A radical partial cystectomy was subsequently performed because of a positive and involved margin. This tumor may have originated from the bladder mucosa and then replaced by intestinal metaplastic cells that originated from the same initiating event.

Appendiceal neoplasms derived from appendiceal tip remnants following appendectomy: a report of two cases.

BACKGROUND: Neoplasms derived from remnant appendix are rarely described, with most cases arising from the appendiceal "stump". Here, we present two surgical cases of appendiceal neoplasms derived from appendiceal "tip" remnants. CASE PRESENTATION: The first patient was a 71-year-old man who had undergone laparoscopic appendectomy for acute appendicitis 12 years prior. During appendectomy, the appendiceal root was ligated, but the appendix was not completely removed due to severe inflammation. At the most recent presentation, computed tomography (CT) was performed to examine choledocholithiasis, which incidentally revealed a cystic lesion of approximately 90 mm adjacent to the cecum. A retrospective review revealed that the cystic lesion had increased in size over time, and laparoscopic ileocecal resection was performed. Pathology revealed no continuity from the appendiceal orifice to the cyst, and a diagnosis of low-grade appendiceal mucinous neoplasm (LAMN) was made from the appendiceal tip remnant. The patient was discharged without complications. The second patient was a 65-year-old man who had undergone surgery for peritonitis due to severe appendicitis 21 years prior. During this operation, the appendix could not be clearly identified due to severe inflammation; consequently, cecal resection was performed. He was referred to our department with a chief complaint of general fatigue and loss of appetite and a cystic lesion of approximately 85 mm close to the cecum that had increased over time. CT showed irregular wall thickening, and malignancy could not be ruled out; therefore, laparoscopic ileocecal resection with D3 lymph node dissection was performed. The pathological diagnosis revealed mucinous adenocarcinoma (TXN0M0) arising from the remnant appendiceal tip. The patient is undergoing follow-up without postoperative adjuvant chemotherapy, with no evidence of pseudomyxoma peritonei or cancer recurrence for 32 months postoperatively. CONCLUSIONS: If appendicitis-associated inflammation is sufficiently severe that accurate identification of the appendix is difficult, it may remain on the apical side of the appendix, even if the root of the appendix is ligated and removed. If the appendectomy is terminated incompletely, it is necessary to check for the presence of a residual appendix postoperatively and provide appropriate follow-up.

Midesophageal diverticulum with elevated intrabolus pressure: a case report.

BACKGROUND: Esophageal diverticulum is commonly associated with esophageal motility disorders, which can be diagnosed using high-resolution manometry (HRM) according to the Chicago classification. Although midesophageal diverticulum (M-ED) is associated with inflammatory processes, esophageal motility disorders have been recently identified as an etiology of M-ED. CASE PRESENTATION: We present the case of a patient with M-ED and elevated intrabolus pressure (IBP), which did not meet the criteria for esophageal motility disorders according to the Chicago classification. A 71-year-old man presented with gradually worsening dysphagia for two years and was diagnosed as having an 8-cm-long M-ED and multiple small diverticula in lower esophagus. HRM revealed a median integrated relaxation pressure of 14.6 mmHg, a distal latency of 6.4 s, and an average maximum IBP of 35.7 mmHg. He underwent thoracoscopic resection of the M-ED and myotomy, which successfully alleviated the symptoms and reduced the intrabolus pressure to normal levels. CONCLUSIONS: It is important to recognize the esophageal diverticulum pathology with HRM findings even in cases where the results may not meet the Chicago classification and to include myotomy based on the results.

Total gastrectomy as a risk factor for postoperative loss of skeletal muscle in minimally invasive surgery for patients with gastric cancer.

PURPOSE: Loss of skeletal muscle mass after gastrectomy for gastric cancer leads to decreased quality of life and poor postoperative survival. However, few studies have examined the postoperative loss of skeletal muscle mass following minimally invasive gastrectomy. This study investigated the impact of minimally invasive total gastrectomy (MI-TG) on changes in skeletal muscle mass during the early postoperative period. METHODS: Patients who underwent MI-TG or minimally invasive distal or proximal gastrectomy (MI-nonTG) for cStage I-III gastric cancer were retrospectively analyzed (n = 58 vs. 182). Their body composition was measured before surgery and 2 months after surgery. Multivariable linear regression analysis was performed to clarify the impact of the surgical procedure on skeletal muscle index changes using clinically relevant covariates. RESULTS: Skeletal muscle mass decreased more in the MI-TG group than in the MI-nonTG group (median [interquartile range]; -5.9% [-10.6, -3.7] vs -4.5% [-7.3, -1.9], P = 0.004). In multivariable linear regression analysis using clinically relevant covariates, MI-TG was an independent risk factor for postoperative loss of skeletal muscle mass (coefficient - 2.6%, 95% CI -4.5 to -0.68, P = 0.008). CONCLUSIONS: Total gastrectomy was a risk factor for loss of skeletal muscle mass during the early postoperative period. If oncologically feasible, proximal or distal gastrectomy with a small remnant stomach should be considered.

Total pelvic exenteration for mucinous adenocarcinoma arising from an implantation cyst 26 years after surgery for rectal cancer.

A 47-year-old man underwent low anterior resection for rectal cancer and was surveilled for 5 years without metastasis. Twenty-four years later, the patient developed an implantation cyst at the anastomotic site. Two years after the diagnosis, colonoscopy revealed a disintegrated area in the lesion, and pathological examination of the biopsy specimen revealed adenocarcinoma. Due to the suspicion of invasion into the surrounding organs, the patient underwent laparoscopic total pelvic exenteration after neoadjuvant chemoradiotherapy. A transabdominal and transperineal endoscopic approach was used for safe en bloc excision of the tumor. Pathological examination of the specimen confirmed mucinous adenocarcinoma arising from the implantation cyst. Although an implantation cyst is considered benign, it is important to suspect malignant transformation when its appearance changes. For the accurate diagnosis of implantation cysts, surgeons, endoscopists, and radiologists should be aware of this disease.

Downregulation of osteoprotegerin in colorectal cancer cells promotes liver metastasis via activating tumor-associated macrophage.

Osteoprotegerin (OPG) is a secreted cytokine that functions as a decoy receptor for receptor activator of nuclear factor kappa-B (RANK) ligand (RANKL). Anti-RANKL treatment for bone metastasis has been widely accepted for solid tumors. However, the mechanism of OPG-RANKL-RANK signaling in systemic colorectal cancer (CRC) metastasis remains unclear. In this study, we investigated the relevance and function of OPG expression in CRC liver metastasis. First, we performed in silico analysis using The Cancer Genome Atlas public database and found that lower OPG expression in CRC was associated with poor overall survival. Immunohistochemistry analyses using resected specimen from patients with CRC in our institute confirmed the result. Patient-matched primary CRC and liver metastases showed a significant downregulation of OPG expression in metastatic lesions. In CRC cell lines, OPG expression did not suppress cell proliferation and migration. However, OPG expression inhibited macrophage migration by suppressing the RANKL-RANK pathway. Moreover, in vivo mouse liver metastasis models showed that OPG expression in CRC cells suppressed liver metastases. In addition, treatment with an anti-RANKL neutralizing antibody also suppressed liver metastases. These results showed that downregulation of OPG expression in CRC cells promotes liver metastasis by activating tumor-associated macrophage, which can become a candidate for targeted therapy with anti-RANKL neutralizing antibody for CRC liver metastasis.

[A case of unresectable hilar cholangiocarcinoma successfully treated by gemcitabine and S-1 combination chemotherapy].

A 52-year-old male was presented with obstructive jaundice and liver dysfunction. He was diagnosed as hilar cholangiocarcinoma involving the confluence of the right and left hepatic duct and bifurcation of the main portal vein trunk. Swollen lymph nodes in the hepatoduodenal ligament were also detected. ERBD tubes were placed in each B2, 3, and 5 branch. GEM and S-1 combination chemotherapy was carried out for four months. As a reduction in the primary tumor and lymph nodes was observed on CT scan surgical exploration was conducted, and an extended left hepatectomy with partial resection of the portal vein and regional lymph node dissection was achieved. The postoperative course was uneventful, and the patient remained free of recurrence, 34 months after the original diagnosis was made, and 29 months after surgical resection. Thus, GEM and S-1 combination chemotherapy is one of the options for the management of advanced hilar cholangiocarcinoma.

[A long survival of a patient with poor performance status who suffered from advanced right breast cancer with multiple lung metastases controlled by trastuzumab as a key drug].

We report a case of a 60-year-old woman with poor performance status (PS). She suffered from advanced right breast cancer with multiple lung metastases, which was controlled by chemotherapy with trastuzumab as the key drug. The patient presented with a 4 cm-sized large right breast mass. Her PS was poor due to progressive spinocerebellar degeneration. The biopsy specimen of the breast mass showed scirrhous type of the invasive ductal carcinoma (ER+, HER2 2+). Multiple lung metastases were also detected by computed tomography. Considering her poor PS, the patient was treated with mild systemic therapy using trastuzumab as the key drug. A different drug response was achieved between the breast mass and lung metastatic lesions, and the tumors were maintained as stable disease (SD) during first 18 months. However, she finally passed away due to respiratory failure resulting from lung metastasis, 33 months after starting treatment. The autopsy findings showed a difference of HER2 expression between the breast tumor and lung metastatic lesions. It must be recognized that differences of HER2 expression between the primary tumor and metastatic lesions are sometimes demonstrated in patients with breast cancer, and that trastuzumab can be used as a key drug in some patients as in the current case.

Targeting Asparagine Synthetase in Tumorgenicity Using Patient-Derived Tumor-Initiating Cells.

Reprogramming of energy metabolism is regarded as one of the hallmarks of cancer; in particular, oncogenic RAS has been shown to be a critical regulator of cancer metabolism. Recently, asparagine metabolism has been heavily investigated as a novel target for cancer treatment. For example, Knott et al. showed that asparagine bioavailability governs metastasis in a breast cancer model. Gwinn et al. reported the therapeutic vulnerability of asparagine biosynthesis in KRAS-driven non-small cell lung cancer. We previously reported that KRAS-mutated CRC cells can adapt to glutamine depletion through upregulation of asparagine synthetase (ASNS), an enzyme that synthesizes asparagine from aspartate. In our previous study, we assessed the efficacy of asparagine depletion using human cancer cell lines. In the present study, we evaluated the clinical relevance of asparagine depletion using a novel patient-derived spheroid xenograft (PDSX) mouse model. First, we examined ASNS expression in 38 spheroid lines and found that 12 lines (12/37, 32.4%) displayed high ASNS expression, whereas 26 lines (25/37, 67.6%) showed no ASNS expression. Next, to determine the role of asparagine metabolism in tumor growth, we established ASNS-knockdown spheroid lines using lentiviral short hairpin RNA constructs targeting ASNS. An in vitro cell proliferation assay demonstrated a significant decrease in cell proliferation upon asparagine depletion in the ASNS-knockdown spheroid lines, and this was not observed in the control spheroids lines. In addition, we examined asparagine inhibition with the anti-leukemia drug L-asparaginase (L-Asp) and observed a considerable reduction in cell proliferation at a low concentration (0.1 U/mL) in the ASNS-knockdown spheroid lines, whereas it exhibited limited inhibition of control spheroid lines at the same concentration. Finally, we used the PDSX model to assess the effects of asparagine depletion on tumor growth in vivo. The nude mice injected with ASNS-knockdown or control spheroid lines were administered with L-Asp once a day for 28 days. Surprisingly, in mice injected with ASNS-knockdown spheroids, the administration of L-Asp dramatically inhibited tumor engraftment. On the other hands, in mice injected with control spheroids, the administration of L-Asp had no effect on tumor growth inhibition at all. These results suggest that ASNS inhibition could be critical in targeting asparagine metabolism in cancers.

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.

Undifferentiated carcinoma of the common bile duct with intraductal tumor thrombi: report of a case.

We report a case of undifferentiated carcinoma of the common bile duct with intraductal tumor thrombi. A 73-year-old man presented with general malaise. Abdominal computed tomography and magnetic resonance imaging revealed a mass in the distal common bile duct, accompanied by dilatation of the intra- and extrahepatic bile ducts. The patient underwent pancreaticoduodenectomy with regional lymphadenectomy. Gross examination revealed that the distal common bile duct was obstructed by an elastic hard mass, 3.2 × 2.6 cm, accompanied by intraductal tumor thrombi. Microscopically, the nodule was well defined and composed of atypical large tumor cells with bizarre nuclei and little cytoplasm. Immunohistochemically, the tumor cells were diffusely positive for cytokeratin-7 and CAM5.2, but negative for CD56, chromogranin A, and synaptophysin. Thus, a histological diagnosis of undifferentiated carcinoma of the common bile duct was made. The patient recovered uneventfully and has remained free of any signs of recurrence for 18 months since the operation. Undifferentiated carcinomas of the extrahepatic bile duct can be detected early, with the chance of a good prognosis; however, because their biologic growth behavior is still considered aggressive, careful observation after surgery and the initiation of multidisciplinary treatment against recurrence are necessary.

Dual blockade of macropinocytosis and asparagine bioavailability shows synergistic anti-tumor effects on KRAS-mutant colorectal cancer.

Mutations of KRAS gene are found in various types of cancer, including colorectal cancer (CRC). Despite intense efforts, no pharmacological approaches are expected to be effective against KRAS-mutant cancers. Macropinocytosis is an evolutionarily conserved actin-dependent endocytic process that internalizes extracellular fluids into large vesicles called macropinosomes. Recent studies have revealed macropinocytosis's important role in metabolic adaptation to nutrient stress in cancer cells harboring KRAS mutations. Here we showed that KRAS-mutant CRC cells enhanced macropinocytosis for tumor growth under nutrient-depleted conditions. We also demonstrated that activation of Rac1 and phosphoinositide 3-kinase were involved in macropinocytosis of KRAS-mutant CRC cells. Furthermore, we found that macropinocytosis was closely correlated with asparagine metabolism. In KRAS-mutant CRC cells engineered with knockdown of asparagine synthetase, macropinocytosis was accelerated under glutamine-depleted condition, and albumin addition could restore the glutamine depletion-induced growth suppression by recovering the intracellular asparagine level. Finally, we discovered that the combination of macropinocytosis inhibition and asparagine depletion dramatically suppressed the tumor growth of KRAS-mutant CRC cells in vivo. These results indicate that dual blockade of macropinocytosis and asparagine bioavailability could be a novel therapeutic strategy for KRAS-mutant cancers.

Chemosensitivity of Patient-Derived Cancer Stem Cells Identifies Colorectal Cancer Patients with Potential Benefit from FGFR Inhibitor Therapy.

Some colorectal cancer patients harboring FGFR (fibroblast growth factor receptor) genetic alterations, such as copy number gain, mutation, and/or mRNA overexpression, were selected for enrollment in several recent clinical trials of FGFR inhibitor, because these genetic alterations were preclinically reported to be associated with FGFR inhibitor sensitivity as well as poor prognosis, invasiveness, and/or metastatic potential. However, few enrolled patients were responsive to FGFR inhibitors. Thus, practical strategies are eagerly awaited that can stratify patients for the subset that potentially responds to FGFR inhibitor chemotherapy. In the present study, we evaluated the sensitivity to FGFR inhibitor erdafitinib on 25 patient-derived tumor-initiating cell (TIC) spheroid lines carrying wild-type RAS and RAF genes, both in vitro and in vivo. Then, we assessed possible correlations between the sensitivity and the genetic/genomic data of the spheroid lines tested. Upon their exposure to erdafitinib, seven lines (7/25, 28%) responded significantly. Normal colonic epithelial stem cells were unaffected by the inhibitors. Moreover, the combination of erdafitinib with EGFR inhibitor erlotinib showed stronger growth inhibition than either drug alone, as efficacy was observed in 21 lines (84%) including 14 (56%) that were insensitive to erdafitinib alone. The in vitro erdafitinib response was accurately reflected on mouse xenografts of TIC spheroid lines. However, we found little correlation between their genetic/genomic alterations of TIC spheroids and the sensitivity to the FGFR inhibitor. Accordingly, we propose that direct testing of the patient-derived spheroids in vitro is one of the most reliable personalized methods in FGFR-inhibitor therapy of colorectal cancer patients.

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

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.

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.