Abstract 1591: Identification and characterization of surface major vault protein on circulating tumor cells in patients with hepatocellular carcinoma

The prognosis of patients with hepatocellular carcinoma (HCC) is poor because of frequent metastasis and resistance to chemotherapy. Circulating tumor cells (CTCs) in blood have attracted attention as potential seeds for metastasis and an important indicator of treatment outcome. However, the biological properties of CTCs are largely unknown due to rarity and lack of CTC-specific surface markers. Previously, we produced monoclonal antibodies (MAbs) against surface molecules on human embryonic stem cells. We found that some of them bound to surface antigens on HCC cell lines but not to normal primary cells. MAb 63-B6, one of the MAbs, recognized CD45-negative CTCs in 6 out of 8 patients with HCCs, suggesting that 63-B6-reactive antigen may be a novel surface marker on CTCs. In a subsequent study, we found that 63-B6 recognized major vault protein (MVP) on the surface of cancer cells, although MVP has been known as a cytoplasmic and nuclear protein. To investigate the role of surface MVP on HCC cell lines, MVP expression was knocked down in Huh7 cells by small interfering RNA. MVP depletion decreased cell growth and increased apoptotic cell death. When Huh7 cells were treated with a polyclonal anti-MVP antibody recognizing surface MVP, Huh7 cell proliferation was decreased without apoptotic cell death, suggesting that surface MVP regulates cell proliferation. Huh7 cells treated with 63-B6 or the anti-MVP antibodies also inhibited Huh7 cell invasion and migration in vitro. Thus, surface MVP is positively associated with HCC cell proliferation, invasion and migration. To further dissect the functional role of surface MVP during HCC metastasis, blood samples from 60 HCC patients and 6 normal adults were stained with 63-B6, HSA, and/or EMT-related antibodies after the depletion of red blood cells and CD45-positive cells. CTCs were detected with 63-B6 in approximately 92% (≥2 CTCs) of patients, and the cell count measured in 7.5 ml of blood ranged between 2 and 1032. Double staining showed that 80% of HSA-positive CTCs was 63-B6-positive. Seventy-six% of EpCAM-positive CTCs was 63-B6-positive while 95% of vimentin-positive CTCs was 63-B6-positive, suggesting that 63-B6-reactive-surface MVP is more expressed on mesenchymal-phenotypic CTCs. Triple staining further showed that 36% of 63-B6-positive CTCs was EpCAM(-)Vimentin(+) and 19% of 63-B6-positive CTCs was EpCAM(+)Vimentin(+) while 3% of 63-B6-positive CTCs was EpCAM(+)Vimentin(-). Interestingly, 42% of 63-B6-positive CTCs was EpCAM(-)Vimentin(-). These results suggest that 63-B6-reactive surface MVP is a novel surface marker on CTCs in patients with HCCs, where it is expressed predominantly on mesenchymal phenotypic and EpCAM(-)Vimentin(-) CTCs. Citation Format: Hyun Min Lee, Jae Won Joh, Hong Seo Choi, Won Tae Kim, Se Ri Seo, Min Kyu Kim, Hee Jin Chang, Dae Shick Kim, Chun Jeih Ryu. Identification and characterization of surface major vault protein on circulating tumor cells in patients with hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1591.