RERF-LC-AI

CLDNs in Human Squamous Carcinoma RERF-LC-AI Cells

Abnormal expression of claudin (CLDN) subtypes has been reported in various solid cancers. The expression levels of CLDN1, 3-5, 7, 12, and 18 were examined in RERF-LC-AI and LK-2 derived from human squamous carcinoma by real-time PCR analysis. In RERF-LC-AI cells, the expression levels of CLDN1 were higher than those in normal tissues, whereas those of CLDN3-5, 7, and 18 were lower (Fig. 1). On the other hand, the expression levels of CLDN7 and 12 in LK-2 cells were higher than those in normal tissues, whereas those of CLDN3, 4, and 18 were lower. RERF-LC-AI cells were used in experiments that followed because the expression pattern of CLDN subtypes in the cells is similar to that in squamous carcinoma tissues.

FLAG-tagged CLDNs were transfected into RERF-LC-AI cells and the expression was determined using anti-FLAG and anti-CLDN3-5, 7, and 18 antibodies. Each CLDN was expressed in the transfected cells (Fig. 2A). The endogenous expression of CLDN1 was not changed by the expression of CLDN3-5, 7, and 18. Immunofluorescence measurements showed that CLDN3 and 4 are mainly distributed in the cytosol and not colocalized with ZO-1 (Fig. 2B). In contrast, CLDN5, 7, and 18 are colocalized with ZO-1, indicating that these CLDNs are distributed in the tight junctions (TJs).

Fig. 1 Expression profile of CLDNs mRNAs in human squamous carcinoma RERF-LC-AI cells. (Akizuki R, et al., 2016)

Fig. 2 Expression and intracellular localization of exogenous CLDNs in RERF-LC-AI cells. (Akizuki R, et al., 2016)

Establishment of an Ovarian Metastasis Model Using RERF-LC-AI Cells

The incidence of metastatic ovarian tumors has been reported to comprise 7-10% of all ovarian cancers. The common sources of ovarian metastatic tumors are the stomach, colon and rectum, appendix, breast, uterus, lung, and skin (melanoma). Using transvenous inoculation to NOD/SCID mice, the tumorigenicity of the eight human cancer cell lines (MKN‐28, MKN‐45, MKN‐74, TMK‐1, KATO‐III, HGC27, HSKTC, and RERF-LC-AI) was extremely low in our examination. Almost all cell lines did not show any tumorigenicity, including in the lung or liver. Only RERF-LC-AI, however, formed tumors in several organs (Table 1). RERF-LC-AI metastasized to the lung at 100%, which was considered to be a natural result because this cell line was originally derived from lung cancer, and also because the lung should generally be the first organ that cells reach after injection into the tail vein. RERF-LC-AI most frequently metastasized to the ovary (67%) after the lung, and bilateral lesions were observed in one-third of ovarian metastatic cases (Table 1, Fig. 3a). Therefore, it was considered that RERF-LC-AI had a high propensity for ovarian metastasis and that the experimental system of transvenous inoculation of this cell line to NOD/SCID mice could be used as an in vivo model of ovarian metastasis. Histologically, three-quarters of the ovarian tumors in this model demonstrated a stromal reaction, mimicking the sarcomatoid proliferation of ovarian fibroblasts in the Krukenberg tumor (Fig. 3b).

Ovarian involvement was also observed in these six cell lines; however, in the cases of TMK-1 and MKN-28, the ovaries were buried in the disseminated tumors, and the cancer cells were considered to invade the ovaries directly from predominant adjacently disseminated tumors. On the other hand, by inoculation with HGC27, MKN-45, KATO-III, and RERF-LC-AI, significant enlargement of the ovary without adjacent dissemination was observed (Fig. 3a, c). These four cell lines are considered to have some capacity for metastasizing to the ovary.

Table 1. Organ distribution of experimental metastasis after transvenous inoculation of RERF-LC-AI. (Kuwabara Y, et al., 2008)

Fig. 3 Experimental metastasis by inoculation of human carcinoma cell lines. (Kuwabara Y, et al., 2008)