Injectable bio-multifunctional hyaluronic acid-based hydrogels loaded with poly ADP-ribose polymerase inhibitors for ovarian cancer therapy.

The recent use of PARP inhibitors (PARPi) in the maintenance treatment of ovarian tumor has significantly improved the survival rates of cancer patients. However, the current oral administration of PARP inhibitors fails to realize optimal therapeutic effects due to the low bioavailability in cancerous tissues, and often leads to a range of systemic adverse effects including hematologic toxicities, digestive system reactions, and neurotoxicities. Therefore, the demand for an advanced drug delivery system that can ensure effective drug administration while minimizing these unfavorable reactions is pressing. Injectable hydrogel emerges as a promising solution for local administration with the capability of sustainable drug release. In this study, we developed an injectable hydrogel made from aminated hyaluronic acid and aldehyde-functionalized pluronic127 via Schiff base reaction. This hydrogel exhibits excellent injectability with short gelation time and remarkable self-healing ability, and is applied to load niraparib. The drug-loaded hydrogel (HP@Nir hydrogel) releases drugs sustainably as tested in vitro as well as displays significant anti-proliferation and anti-migratory properties on human epithelial ovarian cancer cell line. Notably, HP@Nir hydrogel effectively suppresses the growth of ovarian cancer, without significant adverse reactions as demonstrated in animal studies. Additionally, the developed hydrogel is gradually degraded in vivo for around 20 d, while maintaining good biocompatibility. Overall, the injectable hydrogel loaded with niraparib provides a secure and efficient strategy for the treatment and management of ovarian cancer.

[Retracted] Long non­coding RNA receptor activator of nuclear factor­κB ligand promotes cisplatin resistance in non­small cell lung cancer cells.

[This retracts the article DOI: 10.3892/etm.2021.9949.].

Effects of tetrasodium pyrophosphate coupled with soy protein isolate on the emulsion gel properties of oxidative myofibrillar protein.

The effects of protein oxidation on the emulsion gel properties of myofibrillar protein (MP) in the presence of tetrasodium pyrophosphate (TSPP) and soybean protein isolate (SPI) were investigated from the perspective of interfacial protein interactions. The results showed that the emulsifying activity and emulsion stability of MP increased by 35.2 %-181.6 % with elevated H2O2 concentrations (1-20 mM), while the gel strength and water holding capacity of MP emulsions first increased to a maximum at 5 mM H2O2 and then decreased. TSPP and SPI further reinforced the effects caused by oxidation. The emulsifying properties of MP and its emulsion gel properties were closely related to surface hydrophobicity/hydrogen bonds/hydrophobic interactions and disulfide bonds among interfacial proteins, respectively. However, these correlations became difficult to define when TSPP and SPI were introduced. The study provides a theoretical basis for the strategy development to reduce protein oxidation damage on meat product quality.

Pickering emulsion stabilized by modified pea protein-chitosan composite particles as a new fat substitute improves the quality of pork sausages.

Pickering emulsion is a potential substitute for animal fat due to high stability and solid-like properties. Therefore, the effect of replacing 25%-100% pork backfat with Pickering emulsion (75% corn oil volume fraction) stabilized by modified pea protein-chitosan composite particles on the quality of sausages was studied. All meat pastes exhibited a strong gel-like rheological character (G' > G"). The incorporation of Pickering emulsion in sausages enhanced the textural properties (hardness, springiness, chewiness, cohesiveness and resilience) and the uniformity and compactness of micromorphology, as well as suppressed the cooking loss and TBARS content. In particular, the sausages with a backfat substitution ratio of 100%, showing a similar overall sensory acceptability to the backfat sausage, revealed the best rheological properties, texture properties and micromorphology and the lowest cooking loss and fat oxidation (P < 0.05). The results showed that Pickering emulsion stabilized by modified pea protein-chitosan composite particles is a potential fat substitute for meat products with the desirable characteristics.

Effectiveness of two extended-release buprenorphine formulations during postoperative period in neonatal rats.

Information on the effectiveness of a new long-lasting buprenorphine formulation, extended-release buprenorphine, in the neonatal rat is very limited. This study compares whether a high dose of extended-release buprenorphine (XR-Hi) attenuates thermal hypersensitivity for a longer period than a low dose of extended-release buprenorphine (XR-Lo) in a neonatal rat incisional pain model. Two experiments were performed. Experiment one: Male and female postnatal day-5 rat pups (n = 38) were randomly assigned to 1 of 4 treatment groups and received a subcutaneous administration of one of the following: 1) 0.9%NaCl (Saline), 0.1 mL; 2) sustained release buprenorphine (Bup-SR), 1 mg/kg; 3) XR-Lo, 0.65 mg/kg; and 4) XR-Hi, 1.3 mg/kg. Pups were anesthetized with sevoflurane in 100% O2 and a 5 mm long skin incision was made over the left lateral thigh and underlying muscle dissected. The skin was closed with surgical tissue glue. Thermal hypersensitivity testing (using a laser diode) and clinical observations were conducted 1 hour (h) prior to surgery and subsequently after 1, 4, 8, 24, 48, 72 h of treatment. Experiment two: The plasma buprenorphine concentration level was evaluated at 1, 4, 8, 24, 48, 72 h on five-day-old rat pups. Plasma buprenorphine concentration for all treatment groups remained above the clinically effective concentration of 1 ng/mL for at least 4 h in the Bup-SR group, 8 h in XR-Lo and 24 h in XR-Hi group with no abnormal clinical observations. This study demonstrates that XR-Hi did not attenuate postoperative thermal hypersensitivity for a longer period than XR-Lo in 5-day-old rats; XR-Hi attenuated postoperative thermal hypersensitivity for up to 4 h while Bup-SR and XR-Lo for at least 8 h in this model.

LINC00511 Knockdown Suppresses Resistance to Cisplatin in Lung Adenocarcinoma by Interacting with miR-182-3p and BIRC5.

We studied the role of long intergenic non-protein coding RNA 00,511 (LINC00511) in lung adenocarcinoma (LUAD), with a specific focus on acquired chemoresistance. LINC00511 expression was higher in responders to cisplatin (DDP, another name for cisplantin) than non-responders, in A549/DDP cells than in parental A549 cells and normal human bronchial epithelial cells (16HBE). LINC00511 knockdown decreased the half maximal inhibitory concentration (IC50) value, suppressed A549/DDP cell viability, but induced apoptosis. LINC00511 bound with miR-182 and increased the expression of baculoviral inhibitor of apoptosis protein (IAP) repeat containing 5 (BIRC5). BIRC5 knockdown mimicked the effects of LINC00511 knockdown on the IC50 value, A549/DDP cell viability, and apoptosis. BIRC5 overexpression negated the effects of LINC00511 knockdown on A549/DDP cells. In vivo, LINC00511 knockdown attenuated the tumorigenesis of A549/DDP cells after DDP injection. These results provide a novel LINC00511/miR-182/BIRC5 paradigm to explain the mechanism of acquired DDP resistance.

Degenerative Osteoarthropathy in Laboratory Housed Xenopus (Silurana) tropicalis.

In this case study, 15 adult laboratory Xenopus (Silurana) tropicalis (7 adult males and 8 adult females) were examined for nodular enlargements of the clawed digits (digits 0, I, II, and III) on the hind feet. Radiographs showed smoothly margined, rounded, peripherally mineralized lesions arising from the distal phalanges of digits 0-III with osteoproductive and osteolytic components in all frogs. Micro computed tomography (microCT) scans further revealed interphalangeal (IP), metacarpophalangeal (MCP), and metatarsophalangeal (MTP) joint osteoarthritis characterized by periarticular new bone formation, rounded mineral foci both peripherally and centrally within the joints, and more rarely, linear mineralization palmar/plantar to the joints in the flexor tendons. In the nonclawed digits, the shape of the distal phalanx was variably distorted and both subluxation and malangulation of IP joints were identified. Histologically, nodules corresponded to a peripheral rim of mature cortical bone surrounding central adipose tissue, scattered hematopoietic elements, and residual bone of the distal phalanx. Occasionally, the peripheral rim of cortical bone extended proximally to encompass the distal aspect of adjacent phalanx. MCP, MTP and IP joint spaces of most digits exhibited widespread osteoarthritis characterized by periarticular cartilaginous or osseous metaplasia, bony remodeling, and less frequently, granulomatous osteomyelitis. Nutritional analyses of the feed did not indicate imbalances nor were the lesions consistent with metabolic bone disease. The exact etiopathogenesis of these lesions is unknown; however, we hypothesize that the osteoarthritic changes are due to a combination of the frogs' mature age, the unique structure of the Xenopus spp. claw, genetics and biomechanical forces on the digits and distal phalanges of the hind feet.

Long non-coding RNA receptor activator of nuclear factor-κ B ligand promotes cisplatin resistance in non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) is a common malignancy associated with poor clinical outcomes and high mortality rate. The association between NSCLC development and long non-coding RNA (lncRNA) expression remains to be elucidated. The current study investigated the role of a novel lncRNA, receptor activator of nuclear factor-κ B ligand (RANKL), in the resistance of NSCLC to chemotherapy. RANKL expression was assessed via reverse transcription-quantitative PCR, cell death rate was evaluated using flow cytometry and sensitivity of cisplatin (DDP)-resistant A549/DDP cells to chemotherapy was determined using the Cell Counting Kit-8 assay. Western blotting was performed to quantify p53 protein levels. Compared with matched A549 cells, A549/DDP cells exhibited significant upregulation of RANKL expression. Sensitivity of A549/DDP cells to DDP was restored following RANKL knockdown. A549 cells overexpressing RANKL exhibited notably impaired DDP sensitivity compared with controls. Conversely, downregulated RANKL expression triggered cell death and inhibited cell migration via p53 stimulation and phosphatidylinositol 3-kinase/protein kinase B pathway suppression. The current findings indicate that RANKL contributes to DDP resistance in NSCLC and may represent a novel therapeutic target in this malignancy.

lncRNA CCAT2 promotes radiotherapy resistance for human esophageal carcinoma cells via the miR­145/p70S6K1 and p53 pathway.

The long non­coding RNA colon cancer­associated transcript 2 (CCAT2) is abnormally expressed in various types of malignant tumor tissues and considered to be an oncogene, including for esophageal cancer (EC). Radiotherapy is an important and widely used cancer treatment. However, some patients with EC do not respond to radiotherapy. This study was designed to investigate effects of CCAT2 expression on radiotherapy dynamics for EC cells and to explore underlying molecular mechanisms. Reverse transcription­quantitative PCR was used to measure CCAT2 expression in EC tissues, normal esophageal mucosa, EC cells and normal human esophageal epithelial cells. TUNEL assays were used to assess the effect of CCAT2 on X­ray­induced apoptosis of EC cells. Protein expression was detected by western blot. CCAT2 was highly expressed in EC tissues and EC cells, and was negatively associated with radiotherapy efficacy in patients with EC. In vitro, knockdown of CCAT2 enhanced radiosensitivity of EC cells and promoted apoptosis by increasing Bax/Bcl2 and active­caspase 3/caspase 3 following X­ray treatment. In addition, CCAT2 negatively regulated miR­145 and P70 ribosomal protein S6 kinase 1 (p70S6K1) expression, and inhibited phosphorylation of Akt, ERK and p70S6K1 in EC cells. After X­ray treatment, CCAT2 negatively regulated protein levels of p53, P21 and c­Myc. These results showed that CCAT2 promoted the radiotherapy resistance of EC cells via negative regulation of the miR­145/p70S6K1 and the p53 signaling pathways and associated elements may be potential targets for improving the sensitivity of EC radiotherapy.

Comprehensive Transcriptome Reveals an Opposite Regulatory Effect of Plant Growth Retardants in Controlling Seedling Overgrowth between Roots and Shoots.

Seedling overgrowth always develops in undernourished plants due to biotic or abiotic stresses, which significantly decrease the yield of crops and vegetables. It is known that the plant growth retardants paclobutrazol (PBZ) and chlormequat chloride (CCC) are the most commonly used chemicals in controlling seedling height in plants by regulating the gibberellin (GA) biosynthesis pathway. However, the exact molecular regulation mechanism remains largely unknown. This study performed a comprehensive transcriptome profile to identify significantly differentially expressed genes after adding CCC and PBZ to the water culture seedling raising system for the first time. According to the obviously restrained shoots and roots, the GA biosynthesis genes were significantly decreased, as well as the endogenous GA content being reduced. Intriguingly, the GA signaling pathway genes were affected in opposite ways, increasing in roots but decreasing in shoots, especially regarding the phytochrome interacting factor SlPIF1 and the downstream genes expansins (SlEXPs), which promote cell wall remodeling. Further study found that the most down-regulated genes SlEXPA5 and SlEXPA15 were expressed specifically in shoot tissue, performing the function of repressing elongation, while the up-regulated genes SlEXPB2 and SlEXPB8 were proven to be root-specific expressed genes, which may promote horizontal elongation in roots. This research reported the comprehensive transcriptome profiling of plant growth retardants in controlling seedling overgrowth and restraining GA biosynthesis through the regulation of the GA signaling-related genes SlPIF1 and SlEXPs, with an opposite expression pattern between roots and shoots.

Natural and synthetic flavonoids, novel blockers of the volume-regulated anion channels, inhibit endothelial cell proliferation.

Natural flavonoids are ubiquitous in dietary plants and vegetables and have been proposed to have antiviral, antioxidant, cardiovascular protective, and anticancer effects. Volume-regulated anion channels (VRACs), which are essential for cell volume regulation, have been proposed to play a key role in cell proliferation and migration, apoptosis, transepithelial transport, and cancer development. In this study, we screened a group of 53 structurally related natural flavonoids and three synthetic flavonoids for their inhibitory activities on VRAC currents. A whole-cell patch technique was used to record VRAC currents in the human embryonic kidney (HEK) 293 and human umbilical vein endothelial (HUVEC) cells. The 5'-bromo-2-deoxyuridine (BrdU) assay technique was used to investigate cell proliferation. At 100 µM, 34 of 53 compounds significantly inhibited hypotonic extrasolution-induced VRAC currents by > 50% in HEK293 cells. Among these compounds, luteolin, baicalein, eupatorin, galangin, quercetin, fisetin, karanjin, Dh-morin, genistein, irisolidone, and prunetin exhibited the highest efficacy for VRAC blockade (the mean inhibition > 80%) with IC50s of 5-13 µM and Emaxs of about 87-99%. We also studied the effects of three synthetic flavonoids on VRAC currents in HEK293 cells. Flavoxate showed high inhibition efficacy toward VRAC currents (IC50 = 2.3 ± 0.3 µM; Emax = 91.8% ± 2.7%). Finally, these flavonoids inhibited endogenous VRAC currents and cell proliferation in endothelial cells. This study demonstrates that natural and synthetic flavonoids are potent VRAC current inhibitors, and VRAC inhibition by flavonoids might be responsible for their anti-angiogenic effects.

Expression and significance of RRBP1 in esophageal carcinoma.

OBJECTIVE: This study was to investigate the expression and clinical significance of RRBP1 in esophageal carcinoma. MATERIALS AND METHODS: RRBP1 expression was detected in 120 esophageal carcinoma and matched adjacent normal tissues, and the relationship of RRBP1 with clinicopathological characteristics and prognosis was analyzed. RESULTS: RRBP1 was highly expressed in esophageal carcinoma tissues compared with matched adjacent normal tissues (P<0.05). Moreover, RRBP1 expression was associated with T stage, lymph node metastasis, and TNM stage in esophageal carcinoma (P<0.05). Survival analysis revealed that RRBP1, T stage, lymph node metastasis, and TNM stage were significantly associated with patients' prognosis. CONCLUSION: RRBP1 is highly expressed in esophageal carcinoma and can serve as a potential biomarker to predict patients' prognosis.

Setup errors in radiation therapy for thoracic tumor patients of different body mass index.

PURPOSE: To assess the setup errors in radiation therapy for thoracic tumors patients of different somatotypes, and to seek an individualized mathematical basis for defining the planning target volume (PTV). METHODS: Sixty patients with thoracic tumors were divided into four somatotypes according to their body mass index (BMI), and their body positions were setup by two groups of technicians independently. CT simulations were performed and the reconstructed radiography was digitally generated as reference images for location verification and error measurement. By setting positioning error ranges, the within-range positioning correction rate was compared among groups. RESULTS: Position setups for patients in the emaciated group, moderate group, and overweight group were relatively stable (with minor setup error differences between the two groups of technicians). In emaciated group, moderate group, overweight group, and obese group, setup errors in the right-left direction (R-L) were 2.2 ± 1.3 mm, 2.2 ± 1.6 mm, 3.9 ± 3.1 mm, and 8.8 ± 3.5 mm, respectively; whereas the setup errors in the four groups in the superior-inferior(S-I) direction were 2.4 ± 1.8 mm, 2.1 ± 1.9 mm, 3.2 ± 2.6 mm, and 5.4 ± 3.5 mm, and in the anterior-posterior (A-P) direction were 2.2 ± 1.7 mm, 1.9 ± 1.9 mm, 3.2 ± 2.9 mm, and 6.2 ± 4.2 mm, respectively. Moreover, in the moderate group, the positioning correction rate in the three directions (R-L, S-I, and A-P) was 20%, 9%, 8% within the error range of 5-10 mm, and 3%, 0%, 1% with a more than 10 mm error range. However, in overweight group and obese group, the positioning correction rate in these three directions (also with a more than 10 mm error range) was 23%, 27%, 19% and 21%, 16%, 23%, respectively. CONCLUSIONS: In radiation therapy for patients with thoracic tumors, the definition of PTV should be individualized. Meanwhile, with the increase in BMI, positioning correction rate has a tendency to rise too.

The combination of plasma fibrinogen and neutrophil lymphocyte ratio (F-NLR) is a predictive factor in patients with resectable non small cell lung cancer.

The prognostic value of inflammation indexes in non small cell lung cancer (NSCLC) was not established. Therefore, we assessed the clinical applicability of the F-NLR score, which is based on fibrinogen (F) and the neutrophil-lymphocyte ratio (NLR), and the glasgow prognostic score (GPS) to predict the prognoses of NSCLC patients. We retrospectively identified 515 patients with stage I/II/IIIA who underwent surgery at our institution, and evaluated their preoperative serum levels of CRP, albumin, fibrinogen, neutrophil count, and the lymphocyte count. The cut-off values of the fibrinogen level and NLR were determined with receiver operating characteristic (ROC) curve. GPS was classified into three groups as previously described. The disease free survival (DFS) and overall survival (OS) were calculated by the Kaplan-Meier method. Categorical variables were compared using the χ2 test. Survival curves were estimated using the Kaplan-Meier method, and the Cox proportional hazard model was used to assess the prognostic factors. The F-NLR was significantly associated with sex (p = 0.000), smoking history (p = 0.014), lesion type (p = 0.000), histologic type (p = 0.000), T stage (p = 0.000), venous invasion (p = 0.000), lymphatic invasion (p = 0.000), and TNM stage (p = 0.000). The 5-year DFS rates in F-NLR groups 0, 1, and 2 were 46.7%, 36.4%, 30.1%, respectively (p = 0.000), and the 5-year overall survival (OS) rates in the above three groups were 52.0%, 39.8%, 32.1%, respectively (p = 0.000). Multivariate analysis showed that venous invasion (p = 0.036), lymph node metastasis (p = 0.000), and F-NLR (p = 0.034) were independent prognostic factors for DFS. Age (p = 0.015), venous invasion (p = 0.024), lymph node metastasis (p = 0.000), and F-NLR (p = 0.019) were independent prognostic factors for OS. Thus, F-NLR was the independent prognostic factor for both the DFS and OS. And patients with a high-risk preoperative F-NLR group may benefit from adjuvant therapy by subgroup analysis. Our results demonstrated that F-NLR, a novel inflammation-based grading system, as well as the GPS, appeared to have value as a promising clinical predictor of the prognosis for the resectable non small cell lung cancer patients.

Circular RNA expression profiles reveal that hsa_circ_0018289 is up-regulated in cervical cancer and promotes the tumorigenesis.

Circular RNAs (circRNAs) are a type of non-coding RNAs that have been identified as critical regulators in various diseases, especially in cancers. However, the expression profiles and functions of circRNAs in cervical cancer are still unclear. In present study, human circRNAs microarray were performed to screen the circRNAs expression in cervical cancer tissue. Microarray analysis revealed 45 significantly expressed circRNAs with 4 fold change. Among these up-regulated circRNAs, hsa_circ_0018289 was validated to be significantly up-regulated in 35 pairs of cervical cancer tissue compared with adjacent normal tissue and cell lines. Loss-of-function experiments revealed that, in vitro and in vivo, hsa_circ_0018289 knockdown inhibited the proliferation, migration and invasion of cervical cancer cells. Via bioinformatics prediction program and luciferase reporter assays, hsa_circ_0018289 was observed to directly bind to miR-497. Taken together, the results indicate that hsa_circ_0018289 plays important role in cervical cancer proliferation, migration and invasion, suggesting the miRNA 'sponge' of hsa_circ_0018289 and its oncogenic role on cervical cancer tumorigenesis.

MiR-214 inhibits the proliferation and invasion of esophageal squamous cell carcinoma cells by targeting CDC25B.

BACKGROUND: Dysregulation of microRNA(miRNAs) expression was reported in human esophageal squamous cell carcinoma (ESCC). MiR-214 has been found to acts as a tumor suppressor in some tumors including ESCC. The objective of the study was to investigate the functional effect of miR-214 on the regulation of human ESCC progression. METHODS: The expression levels of miR-214 in 57 paired human ESCC tissues and adjacent normal tissues were examined by qRT-PCR. The capacities of cell proliferation and invasion were determined after up-regulation or down-regulation of miR-214 by performing cell viability assay, colony formation assay and transwell assay. Dual luciferase assays, Western blot analysis and qRT-PCR assay were used to demonstrate the association between CDC25B and miR-214. Western blot analysis assessed relative CDC25B protein expression. RESULTS: We observed that miR-214 expression exhibited a frequent down-regulation in ESCC tissues and cells, compared to adjacent normal tissues and cells, respectively. Furthermore, up-regulation of miR-214 significantly inhibited cell proliferation and colony formation and cell invasion capacities in Eca9706 and Eca109 cells. However, down-regulation of miR-214 exhibited an opposite effects. Dual luciferase assays showed that CDC25B was identified as a direct target of miR-214. Meanwhile, up-regulation of miR-214 decreased CDC25B expression, whereas, down-regulation of miR-214 increased the CDC25B expression in Eca9706 and Eca109 cells. Moreover, we demonstrated that miR-214 inhibited Eca9706 and Eca109 cells proliferation and invasion through CDC25B. CONCLUSION: Our results indicate that miR-214 function as a tumor suppressor and may be potential therapeutic target for ESCC.

Long Noncoding RNA PVT1 Facilitates Cervical Cancer Progression via Negative Regulating of miR-424.

Emerging evidence suggests that the long noncoding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) gene is involved in the pathogenesis of cervical cancer. However, the potential mechanism is rarely reported. Our study found that PVT1 was upregulated in cervical cancer tissue and cell lines. After transfecting PVT1 siRNA, the proliferation, migration, and invasion of cervical cancer cells were markedly decreased. miRNA expression profiles demonstrate that miR-424 was markedly downregulated in cervical cancer tissue. Bioinformatics analysis revealed that miR-424 was potentially targeted by PVT1, which was confirmed by dual-luciferase reporter assay. Pearson's correlation analysis showed that PVT1 expression was negatively related to miR-424 expression in glioma cancer tissues. Finally, lowered expression of miR-424 could recover the tumor-suppressive effects of PVT1 knockdown in cervical cancer cell lines. Our results reveal a tumor-promoting role for PVT1, acting as a competing endogenous RNA (ceRNA) or a molecular sponge in negatively modulating miR-424, which might provide a novel therapeutic target for cervical cancer.

Determination of radiotherapeutic target zones for thoracic esophageal squamous cell cancer with lower cervical lymph node metastasis according to CT-images.

Esophageal squamous cell carcinoma (ESCC) is a leading cause of cancer-related deaths worldwide. And radical synchronized chemoradiotherapy has become an important treatment measures for this disease. It is necessary to define the therapeutic target zone based on computer tomography(CT)-images for precise radiotherapy. Therefore, we retrospectively analyzed the regularity of lymph node metastasis in lower cervical section of thoracic esophageal cancer based on CT-images and discussed the range of radiotherapy in supraclavicular zone. The lower cervical lymphatic drainage area was divided into cervical tracheoesophageal groove (CTG), medial supraclavicular zone (MSC zone) and lateral supraclavicular zone (LSC zone) based on CT-images. We found that the rate of lymph node metastasis to medial CTG and MSC zone was relatively high. And rate of lymph node metastasis to the above two zones from middle thoracic section was on an increasing trend with the progress of T stage. Patients at stage T3 and T4 with lymph node metastasis in tracheoesophageal groove in middle thoracic section showed a higher rate of lymph node metastasis in MSC zone. These results demonstrated that the CTG and MSC zone should be clinically included in the supraclavicular target zone for radical radiotherapy, and the T-stage and tumor location should be considered simultaneously.