ITIH5 shows tumor suppressive properties in cervical cancer cells grown as multicellular tumor spheroids.

Cervical cancer (CC) arises from premalignant cervical intraepithelial neoplasia (CIN) induced by a persistent infection with human papillomaviruses. The multi-stepwise disease progression is driven by genetic and epigenetic alterations. Our previous studies demonstrated a clear downregulation of inter-α-trypsin-inhibitor-heavy chain 5 (ITIH5) at mRNA and protein levels in CC compared to CIN2/3 and normal cervical tissue. Initial in vitro functional analyses revealed a suppressive effect of ITIH5 on relevant mechanisms for cancer progression in conventional two dimensional (2D) cell culture model systems. Based on these studies, we aimed to investigate the functional relevance of ITIH5 in multicellular tumor spheroid (MCTS) models, which resemble in vivo tumors more closely. We successfully established CC cell line-derived MCTS using the hanging-drop technique. ITIH5 was ectopically overexpressed in HeLa and SiHa cells and its functional relevance was investigated under three dimensional (3D) culture conditions. We found that ITIH5 re-expression significantly suppressed tumor spheroid growth and spheroid invasiveness of both HeLa and SiHa spheroids. Immunohistochemical (IHC) analyses revealed a significant reduction in Ki-67 cell proliferation index and CAIX-positive areas indicative for hypoxia and acidification. Furthermore, we observed an increase in cPARP-positive cells suggesting a higher rate of apoptosis upon ITIH5 overexpression. An effect of ITIH5 expression on the susceptibility of cervical MCTS towards cytostatic drug treatment was not observed. Collectively, these data uncover pronounced anti-proliferative effects of ITIH5 under 3D cell culture conditions and provide further functional evidence that the downregulation of ITIH5 expression during cervical carcinogenesis could support cancer development.

Human serum alters cell culture behavior and improves spheroid formation in comparison to fetal bovine serum.

BACKGROUND: The use of fetal bovine serum (FBS) as growth supplement for human cell and tissue culture is widely spread in basic research as well as in clinical approaches, although several limitations must be considered, such as unstable composition and availability, biosafety and ethical aspects. Regarding interspecies differences, xenogeneic growth factors may evoke incompatibilities and non-desired interactions with human cells resulting in imprecise outcome of human-relevant data. METHODS: In this study the functionality of human serum (HS) has been investigated in comparison to FBS by assessing proliferation, migration and invasion of the human cervical cancer cell lines SiHa and HeLa. The effects of both sera on spheroid formation were analyzed microscopically. RESULTS: Both, FBS and HS, stimulate cell proliferation and migration similarly, whereas HS significantly enhanced cell invasion. The spheroid formation assay revealed remarkable differences between both sera, especially for SiHa cells. While in FBS supplemented medium cells only formed loose aggregates, HS induced regularly shaped spheroids under all tested conditions. CONCLUSION: We were able to demonstrate that HS and FBS differently influence behavior of cells in culture which may have an impact on experimental results, especially in 3D cultures.