Ki-67 as a Molecular Target for Therapy in an In vitro Three-Dimensional Model for Ovarian Cancer

Abstract

Targeting molecular markers and pathways implicated in cancer cell growth is a promising avenue for developing effective therapies. Although the Ki-67 protein (pKi-67) is a key marker associated with aggressively proliferating cancer cells and poor prognosis, its full potential as a therapeutic target has never before been successfully shown. In this regard, its nuclear localization presents a major hurdle because of the need for intracellular and intranuclear delivery of targeting and therapeutic moieties. Using a liposomally encapsulated construct, we show for the first time the specific delivery of a Ki-67-directed antibody and subsequent lighttriggered death in the human ovarian cancer cell line OVCAR-5. Photoimmunoconjugate-encapsulating liposomes (PICEL) were constructed from anti-pKi-67 antibodies conjugated to fluorescein 5(6)-isothiocyanate, as a photoactivatable agent, followed by encapsulation in noncationic liposomes. Nucleolar localization of the PICELs was confirmed by confocal imaging. Photodynamic activation with PICELs specifically killed pKi-67-positive cancer cells both in monolayer and in three-dimensional (3D) cultures of OVCAR-5 cells, with the antibody TuBB-9 targeting a physiologically active form of pKi-67 but not with MIB-1, directed to a different epitope. This is the first demonstration of (a) the exploitation of Ki-67 as a molecular target for therapy and (b) specific delivery of an antibody to the nucleolus in monolayer cancer cells and in an in vitro 3D model system. In view of the ubiquity of pKi-67 in proliferating cells in cancer and the specificity of targeting in 3D multicellular acini, these findings are promising and the approach merits further investigation.

Impact of treatment response metrics on photodynamic therapy planning and outcomes in a three-dimensional model of ovarian cancer

“…Previous studies from our group have demonstrated that BPD-PDT significantly decreases the size of 3-D OvCa nodules 20 and synergistically enhances carboplatin efficacy. 21 These and other studies 31,59,61,68 suggest a role for 3-D models in improved treatment planning, including for PDT regimens. The myriad of variables that influence PDT outcomes, including the marginal benefit gained from dose escalation, PS concentration, fluence, and the metrics used to evaluate treatment response require further optimization.…”

“…20,21,59,61 Briefly, GFR-Matrigel beds were prepared by pipetting 250 μL of chilled GFR-Matrigel solution onto a 24-well plate. The plate was placed in an incubator and matrigel beds were allowed to gel for 30 min at 37°C.…”

Transcriptome Analysis of Recurrently Deregulated Genes across Multiple Cancers Identifies New Pan-Cancer Biomarkers

“…TOP2A is targeted by etoposide (30). ASNS is targeted in asparginase therapy of acute lymphoblastic leukemia (31), and both MKI67 and MCM2 have been studied as biomarkers (32) and (33) and potential drug targets (34,35). Targeting these genes is likely to bring therapeutic value to many patients as they are recurrently upregulated across many cancer types.…”

A three-dimensional microenvironment alters protein expression and chemosensitivity of epithelial ovarian cancer cells in vitro

“…This is the first large-scale analysis of chemoresponse in EOC cell lines comparing 2D and 3D microenvironments, and the results are consistent with smaller studies. [61][62][63][64][65] Previous studies in other tumor types have also shown resistance to chemotherapy and targeted therapies is enhanced in 3D culture models. 61,63 It is also worth noting that the commonly used chemoresistant/chemosensitive ovarian cancer cell line pairs are generated by culturing cells as monolayers in the presence of drug; the 3D-culturing method used here represents a more physiologically relevant model for the generation of novel drug-resistant lines.…”

“…[61][62][63][64][65] Previous studies in other tumor types have also shown resistance to chemotherapy and targeted therapies is enhanced in 3D culture models. 61,63 It is also worth noting that the commonly used chemoresistant/chemosensitive ovarian cancer cell line pairs are generated by culturing cells as monolayers in the presence of drug; the 3D-culturing method used here represents a more physiologically relevant model for the generation of novel drug-resistant lines. The mechanisms underlying increased resistance in 3D models are not yet well understood, but it is possible that changes in gene expression, the presence of hypoxic zones, and/or reduced penetration of drug into the central areas of MCAs are contributory factors.…”