Claudin-4 (CLDN4) is a major epithelial tight junction protein overexpressed in many cancers to maintain the tumor environment. In this report, we aimed to determine the efficacy of targeting CLDN4 in colorectal cancer (CRC) using an anti-CLDN4 extracellular domain antibody, 4D3. CLDN4 was upregulated in CRC metastatic foci. CLDN4 expression in CRC cells was reduced by upregulation of TNFα, which was induced by Clostridium perfringens enterotoxin produced by gut flora. In a nude mouse liver metastasis model, inhibition of metastasis was increased by combination treatment with 5-fluorouracil (FU) and 4D3 compared to that with 5-FU alone. Moreover, combination treatment with 4D3 and anti-epithelial growth factor receptor (EGFR) antibody C225 resulted in more pronounced inhibition of in vitro sphere formation and tumor growth in nude mice compared to that observed with C225 alone. Moreover, the time interval between the administration of 4D3 and that of C225 was important for maximizing the C225-induced inhibition of EGFR phosphorylation. In a nude mouse model, sequential treatment with 4D3 and C225 with a 6-h time interval resulted in more pronounced inhibition of tumor growth than concurrent treatment. These findings suggest that the targeting of CLDN4 enhances the antitumoral effects of chemotherapeutic agents and molecular targeting antibodies when used in combination.

One of the challenges of using imaging techniques as a tool to study cellular physiology has been the inability to resolve structures that are not located near the surface of the preparation. Nonlinear optical microscopy, in particular two photon-excited fluorescence microscopy (2PM), has overcome this limitation, providing deeper optical penetration (several hundred µm) in ex vivo and in vivo preparations. We have used this approach in the gut to achieve the first in vivo imaging of enteric neurons and nerve fibers in the mucosa, submucosa, submucosal and myenteric plexuses, and circular and longitudinal muscles of the small intestine in H-line: Thy1 promoter GFP mice. Moreover, we obtained clear three-dimensional imaging of enteric neurons that were newly generated after gut transection and reanastomosis. Neurogenesis was promoted by oral application of the 5-HT4-receptor agonist, mosapride citrate (MOS). The number of newly generated neurons observed in mice treated with MOS for one week was 421±89 per 864,900 µm2 (n = 5), which was significantly greater than that observed in preparations treated with MOS plus an antagonist (113±76 per 864,900 µm2) or in 4 week vehicle controls (100±34 per 864,900 µm2) (n = 4 both). Most neurons were located within 100 µm of the surface. These results confirm that activation of enteric neural 5-HT4-receptor by MOS promotes formation of new enteric neurons. We conclude that in vivo 2PM imaging made it possible to perform high-resolution deep imaging of the living mouse whole gut and reveal formation of new enteric neurons promoted by 5-HT4-receptor activation.

| INTRODUC TI ONBreast cancer is the third leading cause of cancer death in Japanese women. 1 The most common histological type, invasive ductal carcinomas (IDC), frequently express hormone receptors (luminal subtype) and/or human epidermal growth factor receptor-2 (HER2, Her2 subtype). 2,3 In contrast, approximately 15% of IDC express none of estrogen receptor (ER), progesterone receptor Abstract Triple negative breast cancer (TNBC) is characterized by highly aggressive phenotype, limited treatment options and a poor prognosis. In the present study, we examined the therapeutic effect of anti-claudin (CLDN)-4 extracellular domain antibody, 4D3, on TNBC. When the expression of CLDN4 and CLDN1 in invasive ductal carcinoma (IDC) was examined in 114 IDC (78 cases from 2004 to 2009 in a single center and 36 cases of tissues array), CLDN1 had lower expression than CLDN4 and was correlated with histological grade. In contrast, expression of CLDN4 was correlated with histological grade, receptor subtype, and stage. CLDN4 expression in human IDC cell lines MCF-7 (luminal subtype) and MDA-468 (TNBC) was at the same level. In both cells, paclitaxel (PTX)-induced growth suppression was enhanced by 4D3. Furthermore, 4D3 increased both intracellular PTX concentration (in both cells) and apoptosis. In the mouse model, 4D3 promoted the antitumor effect of PTX on subcutaneous tumors and reduced lung metastasis. The combination of PTX and 4D3 reduced M2 macrophages and mesenchymal stem cells in the tumor. 4D3 also reduced stemness of the tumors and increased the intratumoral pH. Moreover, concurrent treatment with 4D3, PTX and tamoxifen, or with PTX and tamoxifen in MDA-468 also showed the same level of antitumor activity and survival as MCF-7. Furthermore, in a bone metastasis model, combination of PTX and bisphosphonate with 4D3 promoted tumor growth in both cells. Thus, CLDN4 targeting of the antibody facilitated existing therapeutic effects. K E Y W O R D S breast cancer, chemotherapy, claudin, microenvironment, tight junction | 1841 LUO et aL.

Claudin (CLDN)-4 expression has been associated with malignancy in various cancers. When CLDN4 expression was examined in oral squamous cell carcinoma (OSCC), 22 out of 57 (39%) cases showed immunoreactivity in the nucleus. Nuclear CLDN4-positive cases showed a stronger correlation with cancer progression than the negative cases. Intratumoral anaerobic bacterial DNA examination revealed nuclear CLDN4 expression in 81% of Clostridium perfringens-positive cases. Treatment of human oral squamous cell carcinoma cell lines HSC3 and HSC4 with Clostridium perfringens enterotoxin (CPE), induced CLDN4 nuclear translocation to enhance epithelial-mesenchymal transition (EMT), stemness, cell proliferation and invasive ability. In addition, CPE treatment suppressed phosphorylation of yes-associated protein-1 (YAP1) and promoted YAP1 nuclear translocation, resulting in increased expression of YAP1 target genes; cyclin D1 and connective tissue growth factor. Moreover, it was revealed that the complex of YAP1, CLDN4 and zona occludens-2 (ZO-2) was formed by CPE treatment, further suppressing YAP1 phosphorylation by LATS1 and activating it. Thus YAP activation in OSCC was regarded important in promoting malignant phenotypes. Our research suggested that the control of oral anaerobic bacteria may suppress YAP activation and in turn tumor progression.

Skeletal muscle volume is associated with prognosis of cancer patients. Maintenance of skeletal muscle is an essential concern in cancer treatment. In nutritional intervention, it is important to focus on differences in metabolism between tumor and skeletal muscle. We examined the influence of oral intake of glucose (0%, 10%, 50%) and 2% medium‐chain fatty acid (lauric acid, LAA, C12:0) on tumor growth and skeletal muscle atrophy in mouse peritoneal metastasis models using CT26 mouse colon cancer cells and HT29 human colon cancer cells. After 2 weeks of experimental breeding, skeletal muscle and tumor were removed and analyzed. Glucose intake contributed to prevention of skeletal muscle atrophy in a sugar concentration‐dependent way and also promoted tumor growth. LAA ingestion elevated the level of skeletal muscle protein and suppressed tumor growth by inducing tumor‐selective oxidative stress production. When a combination of glucose and LAA was ingested, skeletal muscle mass increased and tumor growth was suppressed. Our results confirmed that although glucose is an important nutrient for the prevention of skeletal muscle atrophy, it may also foster tumor growth. However, the ingestion of LAA inhibited tumor growth, and its combination with glucose promoted skeletal muscle integrity and function, without stimulating tumor growth. These findings suggest novel strategies for the prevention of skeletal muscle atrophy.