Development of Human Monoclonal Antibody for Claudin-3 Overexpressing Carcinoma Targeting.

Most malignant tumors originate from epithelial tissues in which tight junctions mediate cell-cell interactions. Tight junction proteins, especially claudin-3 (CLDN3), are overexpressed in various cancers. Claudin-3 is exposed externally during tumorigenesis making it a potential biomarker and therapeutic target. However, the development of antibodies against specific CLDN proteins is difficult, because CLDNs are four-transmembrane domain proteins with high homology among CLDN family members and species. Here, we developed a human IgG1 monoclonal antibody (h4G3) against CLDN3 through scFv phage display using CLDN3-overexpressing stable cells and CLDN3-embedded lipoparticles as antigens. The h4G3 recognized the native conformation of human and mouse CLDN3 without cross-reactivity to other CLDNs. The binding kinetics of h4G3 demonstrated a sub-nanomolar affinity for CLDN3 expressed on the cell surface. The h4G3 showed antibody-dependent cellular cytotoxicity (ADCC) according to CLDN3 expression levels in various cancer cells by the activation of FcγRIIIa (CD16a). The biodistribution of h4G3 was analyzed by intravenous injection of fluorescence-conjugated h4G3 which showed that it localized to the tumor site in xenograft mice bearing CLDN3-expressing tumors. These results indicate that h4G3 recognizes CLDN3 specifically, suggesting its value for cancer diagnosis, antibody-drug conjugates, and potentially as a chimeric antigen receptor (CAR) for CLDN3-expressing pan-carcinoma.

Enhanced Cell Growth of Adipocyte-Derived Mesenchymal Stem Cells Using Chemically-Defined Serum-Free Media.

The multipotency and anti-inflammatory effects of mesenchymal stem cells (MSCs) make them attractive for cell therapy in regenerative medicine. A large number of MSCs is required for efficient therapy owing to the low homing efficiency of MSCs to target sites. Furthermore, owing to limitations in obtaining sufficient amounts of MSCs, in vitro expansion of MSCs that preserves their differentiation and proliferative potential is essential. The animal factor included in culture media also limits clinical application. In this study, adipose-derived MSCs showed a significantly higher proliferation rate in STK2, a chemically-defined medium, than in DMEM/FBS. The expression of MSC surface markers was increased in the culture using STK2 compared to that using DMEM/FBS. Tri-lineage differentiation analyses showed that MSCs cultured in STK2 were superior to those cultured in DMEM/FBS. In addition, MSCs cultured in STK2 showed a reduced senescence rate, small and homogenous cell size, and were more genetically stable compared to those cultured in DMEM/FBS. Furthermore, secretome analysis showed that the expression of factors related to proliferation/migration, anti-inflammation, and differentiation were increased in STK2 culture medium compared to DMEM/FBS. Taken together, these results suggest that culture using STK2 medium offers many advantages through which it is possible to obtain safer, superior, and larger numbers of MSCs.

MMP11 and CD2 as novel prognostic factors in hormone receptor-negative, HER2-positive breast cancer.

PURPOSE: More accurate prediction of patient outcome based on molecular subtype is required to identify patients who will benefit from specific treatments. METHODS: We selected novel 16 candidate prognostic genes, including 10 proliferation-related genes (p-genes) and 6 immune response-related genes (i-genes), from the gene list identified in our previous study. We then analyzed the association between their expression, measured by quantitative real-time reverse transcription-PCR in formalin-fixed, paraffin-embedded tissues, and clinical outcome in 819 breast cancer patients according to molecular subtype. RESULTS: The prognostic significance of clinical and gene variables varied according to the molecular subtype. Univariate analysis showed that positive lymph node status was significantly correlated with the increased risk of distant metastasis in all subtypes except the hormone receptor-negative, HER2-positive (HR-/HER2+) subtype. Most p-genes were significantly associated with poor prognosis in patients with the HR+/HER2- subtype, whereas i-genes correlated with a favorable outcome in patients with HR-/HER2+ breast cancer. In HR-/HER2+ breast cancer, four genes (three i-genes BTN3A2, CD2, and TRBC1 and the p-gene MMP11) were significantly associated with distant metastasis-free survival (DMFS). A new prognostic model for HR-/HER2+ breast cancer based on the expression of MMP11 and CD2 was developed and the DMFS for patients in the high-risk group according to our model was significantly lower than that for those in the low-risk group. Multivariate analyses revealed that our risk score is an independent prognostic factor for DMFS. Moreover, C-index showed that our risk score has a superior prognostic performance to traditional clinicopathological factors. CONCLUSIONS: Our new prognostic model for HR-/HER2+ breast cancer provides more accurate information on the risk of distant metastasis than traditional clinical prognostic factors and may be used to identify patients with a good prognosis in this aggressive subtype of breast cancer.

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines.

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.

Human Papillomavirus E6/E7-Specific siRNA Potentiates the Effect of Radiotherapy for Cervical Cancer in Vitro and in Vivo.

The functional inactivation of TP53 and Rb tumor suppressor proteins by the HPV-derived E6 and E7 oncoproteins is likely an important step in cervical carcinogenesis. We have previously shown siRNA technology to selectively silence both E6/E7 oncogenes and demonstrated that the synthetic siRNAs could specifically block its expression in HPV-positive cervical cancer cells. Herein, we investigated the potentiality of E6/E7 siRNA candidates as radiosensitizers of radiotherapy for the human cervical carcinomas. HeLa and SiHa cells were transfected with HPV E6/E7 siRNA; the combined cytotoxic effect of E6/E7 siRNA and radiation was assessed by using the cell viability assay, flow cytometric analysis and the senescence-associated ß-galactosidase (SA-ß-Gal) assay. In addition, we also investigated the effect of combined therapy with irradiation and E6/E7 siRNA intravenous injection in an in vivo xenograft model. Combination therapy with siRNA and irradiation efficiently retarded tumor growth in established tumors of human cervical cancer cell xenografted mice. In addition, the chemically-modified HPV16 and 18 E6/E7 pooled siRNA in combination with irradiation strongly inhibited the growth of cervical cancer cells. Our results indicated that simultaneous inhibition of HPV E6/E7 oncogene expression with radiotherapy can promote potent antitumor activity and radiosensitizing activity in human cervical carcinomas.

The synergistic therapeutic effect of cisplatin with Human papillomavirus E6/E7 short interfering RNA on cervical cancer cell lines in vitro and in vivo.

Human papillomavirus (HPV) types 16 and 18 are the major etiologic factors in the development of cervical epithelial neoplasia. Our study was designed to validate antiviral short interfering RNA (siRNA) targeting the E6 and E7 oncogenes as a potential chemosensitizer of cisplatin (cis-diaminedichloroplatinum II; CDDP) in cervical carcinoma. Specifically, the therapeutic efficacy of combination of CDDP and E6/E7-specific siRNA was assessed in an in vivo cervical cancer xenograft models. The combination of CDDP and E6/E7-specific siRNA had greater efficacy than the combination of CDDP and E6-specific siRNA especially in terms of inducing cellular senescence. Through in vitro and in vivo experiments, the mechanism of synergy between these two treatments was revealed, demonstrating that the combination of E6/E7-specific siRNA and CDDP therapy was significantly superior to either modality alone. In vitro, long-term exposure of HeLa cells to the combination of CDDP and E6/E7-specific siRNA induced apoptosis and cellular senescence. In vivo, E6/E7-specific siRNA potentiated the antitumor efficacy of CDDP via induction of apoptosis, senescence and antiangiogenesis. Our results suggest that E6/E7-specific siRNA may be an effective sensitizer of CDDP chemotherapy in cervical cancer.