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 light-triggered 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.

Figure 1

Schema showing proposed mechanism of nanotechnology mediated sub-cellular antibody delivery and subsequent light inactivation of pKi-67 leading to ovarian cancer cell death. TuBB-9 antibody is conjugated to FITC to yield a photoimmunoconjugate (PIC), which is then encapsulated into non-cationic PEGylated liposomes to provide PIC encapsulating liposomes (PICELs). (MIB-1-FITC conjugates are also encapsulated in liposomes and used as control but are not shown in the scheme). These PICELs are internalized by OVCAR-5 cells and a fraction of the TuBB-9-FITC conjugates are released into the cytoplasm. Within 24 h the conjugates relocalize into the nucleus. Proposed mechanisms for this relocalization are the cotransport of the antibody-FITC-conjugates with the Ki-67 protein, after its synthesis in the cytoplasm, or binding to pKi-67 during mitosis after breakdown of the nuclear envelope. Light irradiation inactivates the Ki-67 protein and is followed by cell death of the ovarian cancer cells.

Figure 2

Confocal microscopy confirms sub-nuclear localization of anti-Ki-67-FITC antibody conjugates and subsequent light irradiation causes ovarian cancer cell death in monolayer cultures. A, Confocal laser-scan images of OVCAR-5 cells 24 h after incubation with TuBB-9-FITC, L-TuBB-9-FITC and L-MIB-1-FITC. The free TuBB-9-FITC antibody-conjugate without liposomal encapsulation is impermeant to the cell membrane (left). Both PICEL constructs L-TuBB-9-FITC (center) and L-MIB-1-FITC (right) deliver the FITC labeled antibody conjugates intracellularly (scale bars, 10 µm), which then localize inside the nucleoli (indicated by arrows). B, Viability of OVCAR-5 cells following irradiation with a 488 nm laser (5 J/cm²) is assessed by standard MTT assay. Only cells that are incubated with L-TuBB-9-FITC show a significant reduction in viability over 72 h post-irradiation. Cells incubated with L-MIB-1-FITC constructs and irradiated with the same energy of light show negligible loss of viability. Negative control samples which consist of cells without any constructs but with light irradiation, cells with L-TuBB-9-FITC constructs but no light or with the free TuBB-9-FITC antibody conjugate show no significant loss of viability.

Figure 3

Multiphoton image of OVCAR-5 3D acini. The larger field of view on the left is a representative sample of 3D acini obtained by differential interference contrast microscopy (scale bar, 100 µm). Magnified area shows a crossection of a single acinus captured by two photon fluorescence microscopy with excitation tuned to 750nm to excite endogenous flavins in OVCAR-5. The autofluorescence image reveals outlines of individual cells to show the 3-dimensional cellular structure of the acinus (scale bar, 10 µm). A z-scan through the acinus from which one section is shown in Figure 3 is attached as a supplemental movie.

Figure 4

OVCAR-5 cells grown in 3D cultures show loss of acinar structure and a progressive increase in cell death following treatment with L-TuBB-9-FITC constructs. A, The pKi-67 expression in 3D cultures is lower than that in monolayer cultures as assessed by flow cytometry in fixed cells. B, Time-lapse images of 3D cultures incubated with L-TuBB-9-FITC constructs and irradiated with 5 J/cm² are shown. The destruction of the 3-D acinar structure is clearly seen 70h after light irradiation (scale bars, 200 µm) C, Normalized live/dead-ratio of 3D OVCAR-5 cells after irradiation with a 488nm laser. The live/dead ratio decreases significantly in cells incubated with L-TuBB-9-FITC 72 h post-irradiation.

Figure 5

Treatment of a non-cancer cell line, human lung fibroblasts cells (MRC-5), shows the specificity of the approach for Ki-67 positive proliferating cells. A, Flow cytometry for Ki-67 status in 80% confluent MRC-5 cells reveals that only a small fraction of cells is pKi-67 positive. B, Viability of MRC-5 cells after irradiation with a 488 nm laser (5 J/cm²) is assessed by standard MTT assay. Confluent cells with low pKi-67 expression show negligible loss of viability following incubation with both anti-pKi-67 liposomal constructs and light irradiation.