Mechanisms of tumor immunosuppressive microenvironment formation in esophageal cancer

As a highly invasive malignancy,esophageal cancer(EC)is a global health issue,and was the eighth most prevalent cancer and the sixth leading cause of cancerrelated death worldwide in 2020.Due to its highly immunogenic nature,emerging immunotherapy approaches,such as immune checkpoint blockade,have demonstrated promising efficacy in treating EC;however,certain limitations and challenges still exist.In addition,tumors may exhibit primary or acquired resistance to immunotherapy in the tumor immune microenvironment(TIME);thus,understanding the TIME is urgent and crucial,especially given the importance of an immunosuppressive microenvironment in tumor progression.The aim of this review was to better elucidate the mechanisms of the suppressive TIME,including cell infiltration,immune cell subsets,cytokines and signaling pathways in the tumor microenvironment of EC patients,as well as the downregulated expression of major histocompatibility complex molecules in tumor cells,to obtain a better understanding of the differences in EC patient responses to immunotherapeutic strategies and accurately predict the efficacy of immunotherapies.Therefore,personalized treatments could be developed to maximize the advantages of immunotherapy.

Tumor-derived DEFB1 induces immune tolerance by inhibiting maturation of dendritic cell and impairing CD8+T cell function in esophageal squamous cell carcinoma

Objective:CD8+T cells are the key effector cells in the anti-tumor immune response.The mechanism underlying the infiltration of CD8+T cells in esophageal squamous cell carcinoma(ESCC)has not been clearly elucidated.Methods:Fresh ESCC tissues were collected and grouped according to the infiltration density of CD8+T cells.After the transcriptome sequencing on these samples and the combined analyses with The Cancer Genome Atlas(TCGA)ESCC data,a secreted protein DEFB1 was selected to explore its potential role in the infiltration of CD8+T cells.Bioinformatics analyses,histological verification and in vitro experiments were then performed.Results:DEFB1 was highly expressed in ESCC,and the high expression of DEFB1 was an independent risk factor for overall survival.Since the up-regulation or down-regulation of DEFB1 did not affect the proliferation,migration and apoptosis of ESCC cells,we speculated that the oncogenic effect of DEFB1 was achieved by regulating microenvironmental characteristics.Bioinformatics analyses suggested that DEFB1 might play a major role in the inflammatory response and anti-tumor immune response,and correlate to the infiltration of immature dendritic cell(imDC)in ESCC.Histological analyses further confirmed that there were less CD8+T cells infiltrated,less CD83+mature DC(mDC)infiltrated and more CD1a+imDC infiltrated in those ESCC samples with high expression of DEFB1.After the treatment with recombinant DEFB1 protein,the maturation of DC was hindered significantly,followed by the impairment of the killing effects of T cells in both 2D and 3D culture in vitro.Conclusions:Tumor-derived DEFB1 can inhibit the maturation of DC and weaken the function of CD8+T cells,accounting for the immune tolerance in ESCC.The role of DEFB1 in ESCC deserves further exploration.

Transcriptome sequencing reveals novel biomarkers and immune cell infiltration in esophageal tumorigenesis

BACKGROUND Esophageal squamous cell carcinoma(ESCC)is one of the most common malignancies worldwide,and its development comprises a multistep process from intraepithelial neoplasia(IN)to carcinoma(CA).However,the critical regulators and underlying molecular mechanisms remain largely unknown.AIM To explore the genes and infiltrating immune cells in the microenvironment that are associated with the multistage progression of ESCC to facilitate diagnosis and early intervention.METHODS A mouse model mimicking the multistage development of ESCC was established by providing warter containing 4-nitroquinoline 1-oxide(4NQO)to C57BL/6 mice.Moreover,we established a control group without 4NQO treatment of mice.Then,transcriptome sequencing was performed for esophageal tissues from patients with different pathological statuses,including low-grade IN(LGIN),high-grade IN(HGIN),and CA,and controlled normal tissue(NOR)samples.Differentially expressed genes(DEGs)were identified in the LGIN,HGIN,and CA groups,and the biological functions of the DEGs were analyzed via Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses.The CIBERSORT algorithm was used to detect the pattern of immune cell infilt-ration.Immunohistochemistry(IHC)was also conducted to validate our results.Finally,the Luminex multiplex cytokine analysis was utilized to measure the serum cytokine levels in the mice.RESULTS Compared with those in the NOR group,a total of 681541,and 840 DEGs were obtained in the LGIN,HGIN,and CA groups,respectively.Using the intersection of the three sets of DEGs,we identified 86 genes as key genes involved in the development of ESCC.Enrichment analysis revealed that these genes were enriched mainly in the keratinization,epidermal cell differentiation,and interleukin(IL)-17 signaling pathways.CIBERSORT analysis revealed that,compared with those in the NOR group,M0 and M1 macrophages in the 4NQO group showed stronger infiltration,which was validated by IHC.Serum cytokine analysis revealed that,compared with those in the NOR group,IL-1βand IL-6 were upregulated,while IL-10 was downregulated in the LGIN,HGIN,and CA groups.Moreover,the expression of the representative key genes,such as S100a8 and Krt6b,was verified in external human samples,and the results of immunohistochemical staining were consistent with the findings in mice.CONCLUSION We identified a set of key genes represented by S100a8 and Krt6b and investigated their potential biological functions.In addition,we found that macrophage infiltration and abnormal alterations in the levels of inflam-mation-associated cytokines,such as IL-1β,IL-6,and IL-10,in the peripheral blood may be closely associated with the development of ESCC.

Study on vasculogenic mimicry in malignant esophageal stromal tumors

AIM: To investigate whether malignant esophageal stromal tumors contain PAS-positive patterned matrix-associated vascular channels, which are lined by tumor cells, but not vascular endothelial cells. That is vasculogenic mimicry (VM) independent of tumor angiogenesis. METHODS: Thirty-six tissue samples of malignant esophageal stromal tumors were analyzed. Tissue sections were stained for Vascular endothelial growth factor (VEGF), CD31 and periodic acid Schiff (PAS). The level of VEGF, the microvascular density (MVD) and the vasculogenic mimicry density (VMD) were determined. RESULTS: PAS-positive patterned matrix-associated vascular channels were detected in 33.3% (12/36) of tumor samples. Within these patterned channels, red blood cells were found. The level of VEGF and the MVD in tumors containing patterned channels were significantly higher than those in tumors not containing patterned channels (P < 0.05). At the same time, the malignant degree of tumors was higher, the proportions of tumors containing patterned channels were not only more, but also in the each kind of tumors containing patterned channels. CONCLUSION: In malignant esophageal stromal tumors, a VM mechanism causes some tumor cells to deform themselves and secrete extracellular matrix; thus, PAS-positive patterned matrix-associated vascular channels appear and supplying blood to the tumors to sustain their growth and metastasis.

Proteomic profiling of fetal esophageal epithelium, esophageal cancer, and tumor-adjacent esophageal epithelium and immunohistochemical characterization of a representative differential protein, PRX6

AIM To understand the molecular mechanism of esophageal cancer development and provide molecular markers for screening high-risk populations and early diagnosis. METHODS Two-dimensional electrophoresis combined with mass spectrometry were adopted to screen differentially expressed proteins in nine cases of fetal esophageal epithelium, eight cases of esophageal cancer, and eight cases of tumor-adjacent normal esophageal epithelium collected from fetuses of different gestational age, or esophageal cancer patients from a high-risk area of esophageal cancer in China. Immunohistochemistry(avidin-biotin-horseradish peroxidase complex method) was used to detect the expression of peroxiredoxin(PRX)6 in 91 cases of esophageal cancer, tumoradjacent normal esophageal tissue, basal cell hyperplasia, dysplasia, and carcinoma in situ, as well as 65 cases of esophageal epithelium from fetuses at a gestational age of 3-9 mo.RESULTS After peptide mass fingerprint analysis and search of protein databases, 21 differential proteins were identified; some of which represent a protein isoform. Varying degrees of expression of PRX6 protein, which was localized mainly in the cytoplasm, were detected in adult and fetal normal esophageal tissues, precancerous lesions, and esophageal cancer. With the progression of esophageal lesions, PRX6 protein expression showed a declining trend(P < 0.05). In fetal epithelium from fetuses at gestational age 3-6 mo, PRX6 protein expression showed a declining trend with age(P < 0.05). PRX6 protein expression was significantly higher in well-differentiated esophageal cancer tissues than in poorly differentiated esophageal cancer tissues(P < 0.05).CONCLUSION Development and progression of esophageal cancer result from interactions of genetic changes(accumulation or superposition). PRX6 protein is associated with fetal esophageal development and cancer differentiation.

Importance of early detection of esophageal cancer before the tumor progresses too much for effective treatment

This editorial comments on an article by Qu et al published in the World Journal of Gastrointestinal Oncology.It focuses on the importance of early detection of esophageal cancer,including recurrence or secondary malignancy after chemoradiotherapy(CRT).Endoscopic resection is the first choice for treatment for esophageal cancer remaining within the mucous membrane,while surgery or radical CRT are treatment options for advanced stages depending on the patient’s general condition and desire.Although these treatments are potentially curative,they are more invasive than endoscopic resection.Early-stage esophageal cancer is often asymptomatic and difficult to detect.Uniform periodic endoscopy is unrealistic.Although less burdensome tests exist,including liquid biopsy and urinary biomarkers,these have not yet been widely used in clinical practice.Early detection is important after radical CRT because the local recurrence rate is higher than that after surgery.However,endoscopic resection or photodynamic therapy is indicated if detected in the early stages,and positive results have been reported.Early detection of esophageal cancer is crucial.Endoscopy is the main diagnostic method;however,new and less burdensome methods should be established to ensure early treatment for patients with esophageal cancer.

Esophageal mesenchymal tumors:Endoscopy,pathology and immunohistochemistry

AIM： To study the endoscopic, pathological and immunoo histochemical features of esophageal mesenchymal tumors. METHODS： Twenty-nine patients diagnosed as esophageal rnysenchymal tumors by electronic endoscopy and endoscopic ultrasound （EUS） were observed under light microscopes, and all tissues were stained by the immunohistochemical method. The expression of CD117, CD34, SMA and desmin were measured by staining intensity of cells and positive cell ratios. RESULTS： Endoscopically, esophageal gastrointestinal stromal tumors （GISTs） and leiomyomas （LMs） had similar appearances, showing submucosal protuberant lesions. They all showed low echo images originated from the muscularis propria or muscularis mucosa on EUS. Endoscopy and EUS could not exactly differentiate esophageal GISTs from LMs. Microscopically, there were two kinds of cells： spindle cell type and epitheloid cell type in esophageal GISTs. Leiomyomas and leiomyosarcornas were only of spindle cell type. One malignancy was found in five cases of esophageal GISTs, and one malignancy in 24 cases of leiomyomas and leiomyosarcomas. Using Fisher＇s exact method, the differences of malignant lesion proportion were not significant between esophageal LMs and GISTs, 1/5 vs 1/24 （P 〉 0.05）. All cases of esophageal GISTs were positive for CD117, and 3 cases were also positive for CD34. The 24 cases of leiomyomas and leiomyosarcomas were all negative for CD117 and CD34. The differences of positive rates of CD117 and CD34 were significant between esophageal GISTs and LMs, 5/5 vs 0/24, 3/5 vs 0/24 （P 〈 0.005）. All leiomyomas and leiomyosarcomas were positive for SMA, and desmin. Among 5 cases of esophageal GISTs, 2 cases were SMA positive, and 1 case was desmin positive. The differences in positive rates and expression intensity of SMA and desmin were significant between esophageal LMs and GISTs, 24/24 vs 2/5, 24/24 vs 115 （P 〈 0.005）. CONCLUSION： The most common esophageal mesenchymal tumors are leiomyomas, and esophageal GISTs are less common. Most of esophageal LMs and GISTs are benign. Endoscopy and EUS are the effective methods to diagnose esophageal mesenchymal tumors and they can provide useful information for the treatment of these tumors. However, they cannot exactly differentiate esophageal GISTs from LMs. Pathological, especially immunohistochemical features are useful to differentiate GISTs from leiomyomas.

Circulating tumor DNA dynamics analysis in a xenograft mouse model with esophageal squamous cell carcinoma

BACKGROUND It remains unclear which factors,such as tumor volume and tumor invasion,influence circulating tumor DNA(ctDNA),and the origin of ctDNA in liquid biopsy is always problematic.To use liquid biopsies clinically,it will be very important to address these questions.AIM To assess the origin of ctDNA,clarify the dynamics of ctDNA levels,assess ctDNA levels by using a xenograft mouse after treatment,and to determine whether tumor volume and invasion are related to ctDNA levels.METHODS Tumor xenotransplants were established by inoculating BALB/c-nu/nu mice with the TE11 cell line.Groups of mice were injected with xenografts at two or four sites and sacrificed at the appropriate time point after xenotransplantation for ctDNA analysis.Analysis of ctDNA was performed by droplet digital PCR,using the human telomerase reverse transcriptase(hTERT)gene.RESULTS Mice given two-site xenografts were sacrificed for ctDNA at week 4 and week 8.No hTERT was detected at week 4,but it was detected at week 8.However,in four-site xenograft mice,hTERT was detected both at week 4 and week 6.These experiments revealed that both tumor invasion and tumor volume were asso ciated with the detection of ctDNA.In resection experiments,hTERT was detected at resection,but had decreased by 6 h,and was no longer detected 1 and 3 d after resection.CONCLUSION We clarified the origin and dynamics of ctDNA,showing that tumor volume is an important factor.We also found that when the tumor was completely resected,ctDNA was absent after one or more days.

Mediastinal tuberculous lymphadenitis presenting as an esophageal intramural tumor: A very rare but important cause for dysphagia

Dysphagia associated with esophageal mechanical obstruction is usually related to malignant esophageal diseases. Benign lesions are rarely a cause for this type of dysphagia, and usually occur either as an intramural tumor or as an extrinsic compression. Mediastinal tuberculous lymphadenitis is rare in adults, and even more rarely causes dysphagia. We report two cases of dysphagia in adult patients caused by mediastinal tuberculous lymphadenitis, presenting radiologicaUy and endoscopically as an esophageal submucosal tumor. Based on the clinical and imaging diagnosis, the patients underwent a right thoracotomy, and excision of the mass attached to and compressing the esophagus. Pathological examination of the specimens showed a chronic granulomatous inflammation with caseous necrosis, which was consistent with tuberculous lymphadenitis.

Uncharted Territory: Frequent Relapsing, Steroid Sensitive Secondary Minimal Change Nephrotic Syndrome Cause by Solid Tumor of the Gastro-Esophageal Junction —(Case Presentation and Review of the Literature)

We reported a biopsy proved case of minimal change nephrotic syndrome in a 72-year-old patient. The minimal change nephrotic syndrome has been steroid sensitive, but the patient had 7 relapses over a span of 5 years. Each time the dose of steroid is tapered, a relapse of the nephrotic syndrome occurred. Eventually, the patient was complaining of dysphagia and difficulty swallowing. Hospital work-up with barium swallow, endoscopy, and CT of the chest, abdomen and pelvis, revealed a focal stenotic lesion with mild to moderate esophageal dysmotility 7/15/2022. A diagnosis of an ulcerating lesion with biopsy confirmed a neuro-endocrine carcinoma of the gastro-esophageal junction was entertained. The CT of the chest/abdomen/pelvis, 7/19/2022, has shown, an esophageal mass of 5.1 × 5.6 × 7 cm of the gastro-esophageal junction with ulceration. No evidence of spread beyond the esophagus and stomach. The histology revealed a poorly differentiated neuroendocrine tumor of the gastro-esophageal junction. The patient underwent several rounds of chemotherapy, radiation, and surgery culminating in tumor control. His nephrotic syndrome was resolved after the tumor has been controlled by surgery and chemotherapy.

Coexistence of gastrointestinal stromal tumor, esophageal and gastric cardia carcinomas

Gastric gastrointestinal stromal tumor (GIST), esophageal squamous cell carcinoma and gastric cardia adenocarcinoma are distinct neoplasms originating from different cell layers; therefore, simultaneous development of such carcinomas is relatively rare. Auxiliary examinations revealed coexistence of esophageal and gastric cardia carcinoma with lymph node metastasis in a 77-year-old man. Intraoperatively, an extraluminal tumor (about 6.0 cm × 5.0 cm × 6.0 cm) at the posterior wall of the gastric body, a tumor (about 2.5 cm × 2.0 cm) in the lower esophagus, and an infiltrative and stenosing tumor (about 1.0 cm × 2.0 cm) in the gastric cardia were detected. Wedge resection for extraluminal gastric tumor, radical esophagectomy for lower esophageal tumor, and cardiac resection with gastroesophageal (supra-aortic arch anastomoses) were performed. Postoperative histological examination showed synchronous occurrence of gastric GIST, esophageal squamous cell carcinoma, and gastric cardia adenocarcinoma. Furthermore, immunohistochemistry indicated strong staining for c-Kit/CD117, Dog-1, Ki-67 and smooth muscle, while expression of S-100 and CD34 was negative.

Endoscopic submucosal dissection for esophageal granular cell tumor using the clutch cutter

Endoscopic submucosal dissection (ESD) with a knife is a technically demanding procedure associated with a high complication rate.The shortcomings of this method are the deficiencies of fixing the knife to the target lesion,and of compressing it.These shortcomings can lead to major complications such as perforation and bleeding.To reduce the risk of complications related to ESD,we developed a new grasping type scissors forceps (Clutch Cutter,Fujifilm,Japan) which can grasp and incise the targeted tissue using an electrosurgical current.Esophagogastroduodenoscopy on a 59-year-old Japanese man revealed a 16mm esophageal submucosal nodule with central depression.Endoscopic ultrasonography demonstrated a hypoechoic solid tumor limited to the submucosa without lymph node involvement.The histologic diagnosis of the specimen obtained by biopsy was granular cell tumor.It was safely and accurately resected without unexpected incision by ESD using the CC.No delayed hemorrhage or perforation occurred.Histological examination confirmed that the granular cell tumor was completely excised with negative resection margin.We report herein a case of esophageal granular cell tumor successfully treated by an ESD technique using the CC.

Monitoring disease progression and treatment efficacy with circulating tumor cells in esophageal squamous cell carcinoma: A case report

This study investigated whether changes in circulating tumor cell(CTC) numbers reflect tumor progression and treatment efficacy in esophageal squamous cell carcinoma(ESCC). A 47-year-old male patient with ESCC is presented in this case study. The patient was evaluated for a series of serum tumor markers and subjected to radiological examinations before and after surgery and during follow-up over the course of five years. In addition, the CTCs in 7.5 m L of peripheral blood were enriched by magneticactivated cell sorting negative selection and identified by immunofluorescence staining. Serum tumor markers remained within normal ranges and were discordant with imaging scans during the follow-up. Initially, one CTC was detected in the peripheral blood sample, and 14 were observed seven days after the operation. After 12 wk, subcutaneous metastases and bone metastases occurred, and the number of CTCs increased to 84. After 48 wk, lung metastases were noted, and the CTC level was 21. At 104 wk, the number of CTCs was 14, and disease recurrence was detected by positron emission tomography-computed tomography. The CTC counts were in accord with the imaging studies at several time points. The additional information provided by CTC enumeration could thus facilitate monitoring of disease status and treatment efficacy and provide support for treatment decisions.

Prognostic value of circulating tumor cells in esophageal cancer

AIM To perform a meta-analysis of the related studies to assess whether circulating tumor cells(CTCs) can be used as a prognostic marker of esophageal cancer.METHODS Pub Med, Embase, Cochrane Library and references in relevant studies were searched to assess the prognostic relevance of CTCs in patients with esophageal cancer. The primary outcome assessed was overall survival(OS). The meta-analysis was performed using the random effects model, with hazard ratio(HR), risk ratio(RR) and 95% confidence intervals(95%CIs) as effect measures.RESULTS Nine eligible studies were included involving a total of 911 esophageal cancer patients. Overall analyses revealed that CTCs-positivity predicted disease progression(HR = 2.77, 95%CI: 1.75-4.40, P < 0.0001) and reduced OS(HR = 2.67, 95%CI: 1.99-3.58, P < 0.00001). Further subgroup analyses demonstrated that CTCs-positive patients also had poor OS in different subsets. Moreover, CTCs-positivity was also significantly associated with TNM stage(RR = 1.48, 95%CI: 1.07-2.06, P = 0.02) and T stage(RR = 1.44, 95%CI: 1.13-1.84, P = 0.003) in esophageal cancer.CONCLUSION Detection of CTCs at baseline indicates poor prognosis in patients with esophageal cancer. However, this finding relies on data from observational studies and is potentially subject to selection bias. Prospective trials are warranted.

mi R-382 functions as a tumor suppressor against esophageal squamous cell carcinoma

AIM To explore the effect of miR-382 on esophageal squamous cell carcinoma (ESCC) in vitro and its possible molecular mechanism. METHODS Eca 109 cells derived from human ESCC and Het-1A cells derived from human normal esophageal epithelium were used. Lentivirus-mediated miR-382 was overexpressed in Eca109 cells. The effect of miR-382 on cell proliferation was evaluated by MTT and colony formation assay. For cell cycle analysis, cells were fixed and stained for 30 min with propidium iodide (PI) staining buffer containing 10 mg/mL PI and 100 mg/mL RNase A, and analyzed by BD FACSCalibur (TM) flow cytometer. For cell apoptosis assay, cells were stained with an Annexin V-FITC/PI Apoptosis Detection Kit according to the manufacturer's instructions and analyzed by a dual-laser flow cytometer. Cell invasion and migration abilities were determined through use of transwell chambers, non-coated or pre-coated with matrigel. Levels of proteins related to cell growth and migration were examined by western blotting. RESULTS Endogenous miR-382 was down-regulated in Eca109 cells compared with Het-1A. Introduction of miR-382 not only significantly inhibited proliferation and colony formation, but also arrested cell cycle at the G2/M phase, as well as promoted apoptosis and autophagy in Eca109 cells. Migration, invasion and epithelial-mesenchymal transition of Eca109 cells were suppressed by overexpressing miR-382. Western blotting results showed that miR-382 inhibited the phosphorylation of mTOR and 4E-BP1. CONCLUSION miR-382 functions as a tumor suppressor against ESCC development and metastasis, and could be considered as a potential drug source for the treatment of ESCC patients.

Esophageal granular cell tumors:Report of 9 cases and a literature review

Esophageal granular cell tumors(GCTs) are rare tumors of the esophagus.We evaluated the clinical and pathologic features of 9 esophageal GCT patients(5 men and 4 women) from our institute and reviewed the related disease literature.Patient age ranged from 25 to 53 years(mean:41 years).All the patients were asymptomatic or presented with non-specific symptoms.Most GCTs occurred in the distal esophagus and were less than 6 mm in diameter.Computational analysis showed that the average gray-scale endoscopic ultrasound images of esophageal GCTs were greater than that of esophageal leiomyomas.Eight patients were treated by endoscopic resection,and 1 patient underwent surgical excision.No post-therapy recurrence or metastasis developed during follow-up(mean:36.4 mo,range:1-72 mo).

Exosomes in esophageal cancer: A review on tumorigenesis,diagnosis and therapeutic potential

Exosomes are nanovesicles secreted from various types of cells and can be isolated from various bodily fluids, such as blood and urine. The number and molecular contents, including proteins and RNA of exosomes, have been shown to reflect their parental cell origins, characteristics and biological behaviors. An increasing number of studies have demonstrated that exosomes play a role in the course of tumorigenesis, diagnosis, treatment and prognosis, although its precise functions in tumors are still unclear. Moreover, owing to a lack of a standard approach, exosomes and its contents have not yet been put into clinical practice successfully. This review aims to summarize the current knowledge on exosomes and its contents in esophageal cancer as well as the current limitations/challenges in its clinical application, which may provide a basis for an all-around understanding of the implementation of exosomes and exosomal contents in the surveillance and therapy of esophageal cancer.

Aberrant methylation of the 3q25 tumor suppressor gene PTX3 in human esophageal squamous cell carcinoma

AIM:To identify the novel methylation-silenced gene pentraxin 3(PTX3) in esophageal squamous cell carcinoma(ESCC).METHODS:PTX3 mRNA expression was examined in six human ESCC cell lines,one human immortalized normal esophageal epithelial cell line,primary ESCC tumor tissue,and paired adjacent nontumor tissue using reverse transcription polymerase chain reaction(RTPCR).Semi-quantitative immunohistochemistry was used to examine cellular localisation and protein levels.Methylation specific PCR and bisulphite genomic sequencing were employed to investigate the methylation of the candidate gene.RESULTS:In the majority of ESCC cell lines,we found that PTX3 expression was down-regulated due to gene promoter hypermethylation,which was further confirmed by bisulphite genomic sequencing.Demethylation treatment with 5-aza-2'-deoxycytidine restored PTX3 mRNA expression in ESCC cell lines.Methylation was more common in tumor tissues(85%) than in adjacent nontumor tissues(25%)(P < 0.01).CONCLUSION:PTX3 is down-regulated through promoter hypermethylation in ESCC,and could potentially serve as a biomarker of ESCC.

Cryopreservation for delayed circulating tumor cell isolation is a valid strategy for prognostic association of circulating tumor cells in gastroesophageal cancer

AIM To demonstrate the feasibility of cryopreservation of peripheral blood mononuclear cells(PBMCs) for prognostic circulating tumor cell(CTC) detection in gastroesophageal cancer.METHODS Using 7.5 m L blood samples collected in EDTA tubes from patients with gastroesopheagal adenocarcinoma, CTCs were isolated by epithelial cell adhesion molecule based immunomagnetic capture using the Iso Flux platform. Paired specimens taken during the same blood draw(n = 15) were used to compare number of CTCs isolated from fresh and cryopreserved PBMCs. Blood samples were processed within 24 h to recover the PBMC fraction, with PBMCs used for fresh analysis immediately processed for CTC isolation. Cryopreservation of PBMCs lasted from 2 wk to 25.2 mo(median 14.6 mo). CTCs isolated from pre-treatment cryopreserved PBMCs(n = 43) were examined for associations with clinicopathological variables and survival outcomes.RESULTS While there was a significant trend to a decrease in CTC numbers associated with cryopreserved specimens(mean number of CTCs 34.4 vs 51.5, P = 0.04), this was predominately in samples with a total CTC count of > 50, with low CTC count samples less affected(P = 0.06). There was no significant association between the duration of cryopreservation and number of CTCs. In cryopreserved PBMCs from patient samples prior to treatment, a high CTC count(> 17) was associated with poorer overall survival(OS)(n = 43, HR = 4.4, 95%CI: 1.7-11.7, P = 0.0013). In multivariate analysis, after controlling for sex, age, stage, ECOG performance status, and primary tumor location, a high CTC count remained significantly associated with a poorer OS(HR = 3.7, 95%CI: 1.2-12.4, P = 0.03). CONCLUSION PBMC cryopreservation for delayed CTC isolation is a valid strategy to assist with sample collection, transporting and processing.

Berberine displays antitumor activity in esophageal cancer cells in vitro

To investigate the effects of berberine on esophageal cancer (EC) cells and its molecular mechanisms. METHODS Human esophageal squamous cell carcinoma cell line KYSE-70 and esophageal adenocarcinoma cell line SKGT4 were used. The effects of berberine on cell proliferation were evaluated using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. For cell cycle progression, KYSE-70 cells were stained with propidium iodide (PI) staining buffer (10 mg/mL PI and 100 mg/mL RNase A) for 30 min and cell cycle was analyzed using a BD FACSCalibur flow cytometer. For apoptosis assay, cells were stained with an Annexin V-FITC/PI apoptosis detection kit. The rate of apoptotic cells was analyzed using a dual laser flow cytometer and estimated using BD ModFit software. Levels of proteins related to cell cycle and apoptosis were examined by western blotting. RESULTS Berberine treatment resulted in growth inhibition of KYSE-70 and SKGT4 cells in a dose-dependent and time-dependent manner. KYSE-70 cells were more susceptible to the inhibitory activities of berberine than SKGT4 cells were. In KYSE-70 cells treated with 50 mu mol/L berberine for 48 h, the number of cells in G2/M phase (25.94% +/- 5.01%) was significantly higher than that in the control group (9.77% +/- 1.28%, P < 0.01), and berberine treatment resulted in p21 upregulation in KYSE-70 cells. Flow cytometric analyses showed that berberine significantly augmented the KYSE-70 apoptotic population at 12 and 24 h post-treatment, when compared with control cells (0.83% vs 43.78% at 12 h, P < 0.05; 0.15% vs 81.86% at 24 h, P < 0.01), and berberine-induced apoptotic effect was stronger at 24 h compared with 12 h. Western blotting showed that berberine inhibited the phosphorylation of Akt, mammalian target of rapamycin and p70S6K, and enhanced AMP-activated protein kinase phosphorylation in a sustained manner. CONCLUSION Berberine is an inhibitor of human EC cell growth and could be considered as a potential drug for the treatment of EC patients.