Fig. 13

An in vitro and in vivo summary of the effects of disruption of TIMP-2 expression by either siRNA or CRISPR/Cas9 in OVCAR5 ovarian cancer cell line. Suppression of TIMP-2 expression by 60–76% in T2-KD cells and gRNA2 cell line resulted in EMT-associated changes (decreased expression of E-cad and MMP-2 with corresponding increased expression of MMP-14, SLUG, SNAIL, VIM, N-cad and TGFβ1). There was also increased proliferation, invasion and chemosensitivity to PTX. In 3D spheroid cultures, gRNA2 cells formed tight, round, and compact cell aggregates, that overexpressed MMP-2, MMP-14, E-Cad, N-Cad and TGFβ1. However, when injected into mice they produced a small tumour burden with no tumours infiltrating peritoneal organs and the mice had higher survival rates compared to control and parental cell lines. On the other hand, in gRNA1, edited by CRISPR/Cas9 for TIMP-2 gene, resulted in the inhibition of TIMP-2 expression by 81–87%. These cells exhibited low MMP-2 expression, and high MMP-14, TWIST1 and SNAIL expression, enhanced proliferation, migration, and invasion compared to control cell lines. However, this cell line was resistant to PTX and in 3D spheroid cultures formed long sheath-like cell aggregates, that had enhanced proliferation and expression of invasion marker, KRT14. Furthermore, when injected into mice gRNA1 cells produced a high tumour burden, which infiltrated peritoneal organs such as liver and pancreas and had similar survival rates when compared to controls. These data suggests that the differences in the ratios of TIMP-2 and MMPs may be critical in controlling the tumorigenic functions of ovarian cancer cells