Efficacy of a Cell-Cycle Decoying Killer Adenovirus on 3-D Gelfoam®-Histoculture and Tumor-Sphere Models of Chemo-Resistant Stomach Carcinomatosis Visualized by FUCCI Imaging

Stomach cancer carcinomatosis peritonitis (SCCP) is a recalcitrant disease. The goal of the present study was to establish an in vitro-in vivo-like imageable model of SCCP to develop cell-cycle-based therapeutics of SCCP. We established 3-D Gelfoam® histoculture and tumor-sphere models of SCCP. FUCCI-expressing MKN-45 stomach cancer cells were transferred to express the fluorescence ubiquinized cell-cycle indicator (FUCCI). FUCCI-expressing MKN-45 cells formed spheres on agarose or on Gelfoam® grew into tumor-like structures with G0/G1 cancer cells in the center and S/G2 cancer cells located in the surface as indicated by FUCCI imaging when the cells fluoresced red or green, respectively. We treated FUCCI-expressing cancer cells forming SCCP tumors in Gelfoam® histoculture with OBP-301, cisplatinum (CDDP), or paclitaxel. CDDP or paclitaxel killed only cycling cancer cells and were ineffective against G1/G2 MKN-45 cells in tumors growing on Gelfoam®. In contrast, the telomerase-dependent adenovirus OBP-301 decoyed the MKN-45 cells in tumors on Gelfoam® to cycle from G0/G1 phase to S/G2 phase and reduced their viability. CDDP- or paclitaxel-treated MKN-45 tumors remained quiescent and did not change in size. In contrast, OB-301 reduced the size of the MKN-45 tumors on Gelfoam®. We examined the cell cycle-related proteins using Western blotting. CDDP increased the expression of p53 and p21 indicating cell cycle arrest. In contrast, OBP-301 decreased the expression of p53 and p21 Furthermore, OBP-301 increased the expression of E2F and pAkt as further indication of cell cycle decoy. This 3-D Gelfoam® histoculture and FUCCI imaging are powerful tools to discover effective therapy of SCCP such as OBP-301.

As described in detail previously [2], osteosarcoma growing on Gelfoam 1 , formed threedimensional tissue-like structures, but did not form structures in monolayer culture and only colonies on Matrigel TM [2,3].
We recently observed that cancer cells in G 0 /G 1 phase in Gelfoam 1 histoculture had more extensive migration than in S/G 2 /M. Upon entry into S/G 2 /M, the cells ceased migrating. After mitosis, when the cells entered G 0 /G 1 , the cells could resume migrating. The migrating cells in G 0 /G 1 were not sensitive to chemotherapy. Fluorescence ubiquination cell cycle indicator (FUCCI) enabled monitoring of the cell cycle phase of each cell in real time as described in detail previously [12,13].
We previously showed with FUCCI imaging that cancer cells in Gelfoam 1 and in tumors in mice had a similar cell-cycle-phase distribution. Only the surface cells in Gelfoam 1 histoculture and tumors was cycling; interior cells of tumors and Gelfoam 1 histocultures were quiescent in G 0 /G 1 . In mono-layer culture, in contrast, cancer cells continuously cycle. In both Gelfoam 1 histoculture and tumors in mice, chemotherapy had similar efficacy, in contrast to cancer cells in mono-layer, which were much more chemo-sensitive as described previously in detail [13][14][15].
In the present report, we determined the efficacy of cell-cycle decoying telomerase-dependent adenovirus OBP-301 on stomach cancer carcinoma growing on Gelfoam 1 histoculture, using FUCCI imaging to monitor cell-cycle changes.

Gelfoam 1 Histoculture
Gelfoam 1 (Pharmacia & Upjohn, Kalamazoo, MI), was cut into 1 cm cubes and placed in 6-well tissue culture plates containing RPMI-1640 medium and placed in a 37°C incubator until the Gelfoam 1 absorbed the medium. FUCCI-expressing cancer cells (1×10 6 ) were then carefully layered on top of the Gelfoam 1 and placed in an incubator for 1 h, after which additional medium was added up to the top of the Gelfoam 1 and then incubated again at 37°C with 5% CO 2 /95% air, as described previously in detail [2, 4-6, 12, 18].

Establishment of FUCCI-Expressing MKN-45 Cells
In order to establish FUCCI-expressing MKN-45 cells, two plasmids were utilized: mKO2-hCdt1 containing an orange-red fluorescent protein and mAG-hGem, containint a green fluorescent protein (Medical and Biological Laboratory, Nagoya, Japan) [19] were sequentially tranfected into MKN-45 cells with the use of Lipofectamine™ LTX (Invitrogen, Carlsbad, CA). After transfection with each plasmid, the cells were cultured for appropriate The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funders provided support in the form of salaries for authors [YU], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of all the authors are articulated in the 'author contributions' section.  periods of time and sorted for the fluorescence color corresponding to plasmid used as described previously in detail [12].

Imaging of FUCCI-Expressing MKN-45 Cells
The OV100 variable magnification fluorescence imaging system (Olympus) was used to acquire macro images as described previously in detail [20]. The FV1000 confocal laser scanning microscope (Olympus) was used to image single FUCCI-expressing cells. The FV1000 contains 473 nm and 559 nm lasers [21]. The 4× objective lens with a 0.20 numerical aperature and the 20× objective lens with a 0.95 numerical aperature were used. Fluoview software (Olympus) was used for image acquisition and Velocity 6.0 version (Perkin Elmer) [15] were used for 3D analysis. The above methods were previously described in detail [21].

Statistical Analysis
The Student's t-test was used for comparison of 2 groups. Data are presented as means ± SD. Significance was determined via a P value of < 0.05 [12].

Cell-cycle Decoy and Killer Efficacy of Telomerase-Dependent Adenovirus OBP-301 in Comparison with Chemotherapy on Tumor Spheres
FUCCI-expressing MKN-45 stomach cancer cells cultured on agarose spontaneously formed spheres and were mostly FUCCI red, indicating they were in G 0 /G 1 phase (Fig 1A). Tumor spheres were treated with OBP-301, cisplatinum (CDDP), or, paclitaxel ( Fig 1B). CDDP-or paclitaxel-treated MKN-45 cells remained in the quiescent G 0 /G 1 state and did not change size compared with control-spheres or spheres before treatment (Fig 1C-1E). In contrast, OBP-301 decoyed the cell cycle of tumor spheres from G 0 /G 1 phase to S/G 2 phase where they expressed FUCCI green and reduced the size of the tumor (Fig 1C-1E). G 0 /G 1 cancer cells in spheres were resistant to chemotherapy but could be decoyed to S/G 2 by OBP-301 which reduced their viability.

Cell-cycle Distribution of MKN-45 Stomach Cancer Cells Forming Tumors on Gelfoam 1
FUCCI-expressing MKN-45 cells on Gelfoam 1 grew into tumor-like structures with the majority of the cells in G 0 /G 1 (Fig 2) when the tumor-like structure matured. The mature tumors or spheres on Gelfoam 1 have the vast majority of their cells in G 0 /G 1 (Figs 1E and 3E and 3F) and thereby look red or yellow. The images are of the whole tumor or sphere, not cross  3A). Only cycling MKN-45-FUCCI cells were sensitive to CDDP and paclitaxel; quiescent G 0 /G 1 MKN-45 cells were resistant to these drugs ( Fig 3B). In contrast, OBP-301 decoyed the MKN-45 cells in the tumor-like structures on Gelfoam 1 to cycle from G 0 /G 1 phase to S/G 2 phase and reduced their viability (Fig 3B and 3C). CDDP-or paclitaxel-treated MKN-45 tumor-like structure remained quiescent and did not change in size ( Fig  3D and 3E). In contrast, OBP-301 reduced the size of the MKN-45 tumor-like structures on Gelfoam 1 (Fig 3D-3F).

OBP-301 Modulates Cell-Cycle Related Gene Expression
We examined the cell cycle-related proteins using Western blotting. CDDP increased the expression of p53 and p21 indicating cell-cycle arrest. In contrast, OBP-301 decreased the expression of p53 and p21 (Fig 4). Furthermore, OBP-301 increased the expression of E2F and pAkt indicating in cell-cycle decoy (Fig 4). The cell cycle decoy effect of OBP-301 is large scale, whereby the cells in untreated mature spheres or Gelfoam 1 tumor are almost all in G 0 /G 1 and almost all of them cycle after OBP-301 infection. The sphere or tumor area is reduced by approximately one half after OBP-301 treatment which is not as great an effect as the extent of decoy. Future experiments will investigate the kinetics of cancer-cell killing after OBP-301 treatment.

Conclusions
SCCP growing as spheres or tumor-like structures on Gelfoam 1 cycle became mostly quiescent as they matured and were thereby resistant to chemotherapy. Telomerase-dependent adenovirus OBP-301 could decoy the quiescent tumor cells in spheres or tumor-like structures in Gelfoam 1 to begin to cycle whereby they lost viability. These results are further demonstration of the power of cell-decoy chemotherapy.