Epithelial-mesenchymal transition (EMT) is a crucial pathological event in cancer, particularly in tumor cell budding and metastasis. Therefore, control of EMT can represent a novel therapeutic strategy in cancer. Here, we introduce an innovative three-dimensional (3D) high-throughput screening (HTS) system that leads to an identification of EMT inhibitors. For the establishment of the novel 3D-HTS system, we chose NanoCulture Plates (NCP) that provided a gel-free micro-patterned scaffold for cells and were independent of other spheroid formation systems using soft-agar. In the NCP-based 3D cell culture system, A549 lung cancer cells migrated, gathered, and then formed multiple spheroids within 7 days. Live cell imaging experiments showed that an established EMT-inducer TGF-β promoted peripheral cells around the core of spheroids to acquire mesenchymal spindle shapes, loss of intercellular adhesion, and migration from the spheroids. Along with such morphological change, EMT-related gene expression signatures were altered, particularly alteration of mRNA levels of ECAD/CDH1, NCAD/CDH2, VIM and ZEB1/TCF8. These EMT-related phenotypic changes were blocked by SB431542, a TGF-βreceptor I (TGFβR1) inhibitor. Inside of the spheroids were highly hypoxic; in contrast, spheroid-derived peripheral migrating cells were normoxic, revealed by visualization and quantification using Hypoxia Probe. Thus, TGF-β-triggered EMT caused spheroid hypoplasia and loss of hypoxia. Spheroid EMT inhibitory (SEMTIN) activity of SB431542 was calculated from fluorescence intensities of the Hypoxia Probe, and then was utilized in a drug screening of EMT-inhibitory small molecule compounds. In a pilot screening, 9 of 1,330 compounds were above the thresholds of the SEMTIN activity and cell viability. Finally, two compounds SB-525334 and SU9516 showed SEMTIN activities in a dose dependent manner. SB-525334 was a known TGFβR1 inhibitor. SU9516 was a cyclin-dependent kinase 2 (CDK2) inhibitor, which we showed also had an EMT-inhibitory activity. The half maximal inhibitory concentration (IC50) of SB-525334 and SU9516 were 0.31 μM and 1.21 μM, respectively, while IC50 of SB431542 was 2.38 μM. Taken together, it was shown that this 3D NCP-based HTS system was useful for screening of EMT-regulatory drugs.

The establishment of various serotypes of Streptococcus mutans was studied serologically in plaque samples collected from labial surfaces of upper primary incisors of 22 infants (starting age, 5 to 13 months) over a period of 30 months. Clinical examinations were also performed. No clear-cut association between the initiation of dental caries and previous detection ofS. mutans was noted. However, all 12 of the infants with caries had S. mutans isolated at some time during the course of this study. The most common serotype isolated at the initial establishment of S. mutans on the tooth surfaces was serotype c, whereas types d, e, and g became established in a few cases. During the test period, changes in the distribution of serotypes of S. mutans were observed in some cases. The initiation of carious lesions could be found in a few cases even when S. mutans comprised about 1% or less of the total streptococcal count of the specimen from the tooth surfaces. Serotype dlg strains tended to develop carious lesions on smooth surfaces, although serotype c was isolated from almost all individuals who developed caries.

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Oral implantation and the cariogenic activity of clinical strains of Streptococcus mutans which had been isolated from Japanese children and labeled with streptomycin-resistance were examined in specific pathogen-free Sprague-Dawley rats. All the seven strains tested were easily implanted and persisted during the experimental period. Extensive carious lesions were produced in rats inoculated with clinical strains of S. mutans belonging to serotypes c, d, e, and f, and maintained on caries-inducing diet #2000. Noninfected rats did not develop dental caries when fed diet #2000. Type d S. mutans preferentially induced smooth surface caries in the rats. Strains of other serotypes primarily developed caries of pit and fissure origin. Caries also developed in rats inoculated with reference S. mutans strains BH-TR and FAlR (type b) that had been maintained in the laboratories for many years. However, the cariogenicity of the laboratory strains was found to have decreased markedly. All three S. sanguis strains could be implanted, but only one strain induced definite fissure caries. Two S. salivarius strains could not be implanted well in the rats and therefore they were not cariogenic. Four different species of lactobacilli also failed to induce dental caries in rats subjected to similar caries test regimen on diet #2000. S. mutans strain MT6R (type c) also induce caries in golden hamsters and ICR mice, but of variable degrees.