Exosome-derived ANXA9 functions as an oncogene in breast cancer.

Breast cancer (BCA) is one of the most prevalent cancers among women. Emerging evidence has revealed that Annexin A-9 (ANXA9) plays a crucial function in the development of some cancers. Notably, ANXA9 has been reported to be a new prognostic biomarker for gastric and colorectal cancers. However, its expression and biological function in BCA have not yet been investigated. Using online bioinformatics tools such as TIMER, GEPIA, HPA, and UALCAN, we predicted ANXA9 expression and its correlation with the clinicopathological characteristics of BCA patients. RT-qPCR and western blot were utilized to measure ANXA9 mRNA and ANXA9 protein expression in BCA patient tissues and cells. BCA-derived exosomes were identified by transmission electron microscopy. Functional assays were employed to evaluate the biological role of ANXA9 in BCA cell proliferation, migration, invasion, and apoptosis. A tumor xenograft in vivo model was utilized to assess the role of ANXA9 in tumor growth in mice. Bioinformatics and functional screening analysis revealed that ANXA9 was highly expressed in BCA patient tissues, with median ANXA9 expression 1.5- to 2-fold higher than in normal tissues (p < 0.05). RT-qPCR confirmed that ANXA9 expression in BCA tissues was around 1.5-fold higher than the adjacent normal tissues (p < 0.001). ANXA9 expression in different subtypes of BCA also showed a difference, and ANXA9 was found to be mostly significantly upregulated in luminal BCA relative to normal tissues or other histological subtypes (p < 0.001). Moreover, ANXA9 expression was elevated in different races, ages, clinical stages, node metastasis status, and menopause status groups relative to the normal group (p < 0.001). Furthermore, ANXA9 was found to be secreted by BCA tissue-derived exosomes and its expression was upregulated 1- to 7-fold in BCA cells treated with exosomes (p < 0.001), while its expression in MCF10A cells was not significantly altered by treatment with exosomes (p > 0.05). ANXA9 silencing induced a significant decrease of around 30% in the colony number of BCA cells (p < 0.01). The number of migrated and invaded BCA cells also decreased by around 65 and 68%, respectively, after silencing ANXA9 (p < 0.01). Tumor size was significantly reduced (nearly half) in the LV-sh-ANXA9 group relative to the LV-NC group in the xenograft model (p < 0.01), suggesting that ANXA9 silencing repressed tumor progression in BCA progression in vitro and in vivo. In conclusion, exosome-derived ANXA9 functions as an oncogene that facilitates the proliferation, migration, and invasiveness of BCA cells and enhances tumor growth in BCA development, which may provide a new prognostic and therapeutic biomarker for BCA patients.

Study on the Heterogeneity of T-DM1 and the Analysis of the Unconjugated Linker Structure under a Stable Conjugation Process.

Trastuzumab emtansine (T-DM1) is a target-specific anticancer antibody-drug conjugate (ADC). In the present study, critical quality attributes for different manufactured products, such as the drug to antibody ratio (DAR), conjugation site, and site conjugation ratio, are similar, which is contrary to the traditional view that conjugation at lysine sites is randomly assigned. To investigate this result, a series samples with different DARs were prepared. Site conjugation ratios of the 27 different conjugation sites (corresponding to 54 potential sites) were analyzed. We found that the correlation coefficients of the 26 site conjugation ratio and the DAR were R 2 > 0.9, and the remaining one was R 2 > 0.7. By comparing three batches of samples with a DAR value of ∼3.3 in a stability study, we found that degradation rates of conjugation sites of the samples incubated at 40 °C were basically the same. These data show that the conjugation ratio and the conjugation stability of each site may remain consistent if the process parameters are stable. LC/MS/MS was used to study the unconjugated linker of the crosslink byproducts produced by a two-step method. We determined four forms of unconjugated linkers: (N-maleimidomethyl) cyclohexane-1-carboxylate (MCC) unconjugated to DM1, hydrolyzed MCC unconjugated to DM1, lys-MCC-lys, and lys-MCC-cys. We believe that the current study can provide an effective guide for the processing of ADCs, control of product quality, and reduction of side reaction products.

Time profile of nimotuzumab for enhancing radiosensitivity of the Eca109 cell line.

The aim of the present study was to investigate the ability of Nimotuzumab to increase radiosensitivity at different delivery times in the mixed cancer cell line Eca109, to determine the optimal delivery time. Cultured Eca109 cells were classified into five groups: Control with no treatment (O group); irradiation without Nimotuzumab treatment (R group); treatment with Nimotuzumab 24 h prior to or after irradiation (24NR or 24RN group, respectively); and Nimotuzumab combined with irradiation simultaneously (NR group). Following cells reaching the logarithmic-growth phase, cell survival after exposure to Nimotuzumab was evaluated using an MTT assay; thereafter, the 50% inhibitory concentration (IC50) of the cell line was calculated. Cell-survival curves were generated using a colony-forming assay. Flow cytometry analysis was used to detect apoptosis rates and cell-cycle distribution. The expression level of epidermal growth factor receptor was measured in Eca109 cells with western blotting. Growth inhibition was only observed 72 h after exposure to Nimotuzumab. The IC50 was 768 µg/ml. At a dose of 0.2 IC50 or 0.3 IC50, the sensitization enhancement ratio of radiosensitivity was highest in the 24NR group. Nimotuzumab enhanced radiation-induced apoptosis in Eca109 cells, with the optimal delivery time at 24 h prior to irradiation (P=0.035). The concentration of Nimotuzumab administered was directly proportional to the increase in radiosensitivity of the cells.

Clinical significance of the expression of EGFR signaling pathway-related proteins in esophageal squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) signaling is an important pathway that is not only involved in the determination of cellular development, but also has significant roles in tumor development and progression. The study aims to examine the expression of EGFR signaling pathway-related proteins (EGFR, c-Fos, and c-erb-B2) in esophageal squamous cell carcinoma and to investigate their relationships with clinical significance. Sixty esophageal squamous carcinoma specimens obtained by fiber esophagoscope were subjected to two-step immunohistochemistry to test the expression of EGFR, c-Fos, and c-erb-B2. EGFR expression was observed in 73.3% of tumors (44/60); positive EGFR expression was significantly correlated with tumor-node-metastasis (TNM) staging, lymph node metastasis, and distant metastasis (P < 0.05). c-Fos expression was found in 85% (51/60) of tumors, and its expression was significantly related to tumor depth and TNM staging (P < 0.05). c-erb-B2 expression was 75% (45/60) in esophageal squamous cell carcinoma (ESCC) specimens, and positive-erb-B2 expression had a significant association with the depth of tumor invasion (P < 0.05). c-Fos expression was significantly and positively correlated with c-erb-B2 (P < 0.05). Overexpression of EGFR, c-Fos, and c-erb-B2 was associated with tumor progression and development. EGFR and c-Fos expression can predict the tumor stage. c-Fos and c-erb-B2 expression can be used to determine the depth of tumor invasion and can also act as a combined prognostic indicator in ESCC.

Growth, clonability, and radiation resistance of esophageal carcinoma-derived stem-like cells.

OBJECTIVE: To separate/enrich tumor stem-like cells from the human esophageal carcinoma cell line OE-19 by using serum-free suspension culture and to identify their biological characteristics and radiation resistance. METHODS: OE-19 cells were cultivated using adherent and suspension culture methods. The tumor stem-like phenotype of CD44 expression was detected using flow cytometry. We examined growth characteristics, cloning capacity in soft agar, and radiation resistance of 2 groups of cells. RESULTS: Suspended cells in serum-free medium formed spheres that were enriched for CD44 expression. CD44 was expressed in 62.5% of suspended cells, but only in 11.7% of adherent cells. The suspended cells had greater capacity for proliferation and colony formation in soft agar than the adherent cells. When the suspended and adherent cells were irradiated at 5 Gy, 10 Gy, or 15 Gy, the proportion of CD44+ suspended cells strongly and weakly positive for CD44 was 77.8%, 66.5%, 57.5%; and 21.7%, 31.6%, 41.4%, respectively. In contrast, the proportion of CD44+ adherent cells strongly positive for CD44 was 18.9%, 14.%, and 9.95%, respectively. When the irradiation dose was increased to 30 Gy, the survival of the suspended and adherent cells was significantly reduced, and viable CD44+ cells were not detected. CONCLUSION: Suspended cell spheres generated from OE-19 esophageal carcinoma cells in serum-free stem medium are enriched in tumor stem-like cells. CD44 may be a marker for these cells.