Time-course imaging of decoy of quiescent MKN-45 cancer cells in tumor-like structures on Gelfoam^® by OBP-301 and their subsequent killing.

<p><b>A.</b> Experimental setup for treatment of MKN-45 tumor-like structures growing in Gelfoam^® with OBP-301, CDDP, or paclitaxel. <b>B.</b> Time-course images of FUCCI-expressing MKN-45 cancer cells forming tumor-like structures on Gelfoam^® treated with OBP-301, CDDP, or paclitaxel. The cells in G₀/G₁, S, or G₂/M phases appear red, yellow, or green fluorescent, respectively. <b>C.</b> Histogram shows the cell cycle phase of FUCCI-expressing MKN-45 cancer cells forming tumor-like structures on Gelfoam^® treated with OBP-301, CDDP, or paclitaxel. <b>D.</b> Representative images of control, OBP-301-, CDDP-, or paclitaxel-treated tumors. <b>E.</b> Bar graphs show the size of viable tumor after each treatment. <b>F.</b> Histogram shows cell-cycle phase of MKM-45 cells in treated and untreated spheres. Scale bars, 100 μm. Data are shown as means ± SD (n = 5). *<i>P</i> < 0.01. The percentage of cells in G₀/ G₁, S, and G₂/M phases are shown.</p

OBP-301 decoys MKN-45 stomach cancer cells in G₁/G₀ to cycle in tumor spheres.

<p><b>A.</b> Representative of FUCCI MKN-45 stomach cancer cells <i>in vitro</i>. <b>B.</b> Experimental setup for treatment of FUCCI-expressing MKN-45 cells in spheres with OBP-301, cisplatinum (CDDP), or paclitaxel. <b>C.</b> Representative images of control, OBP-301-, CDDP- or paclitaxel-treated spheres. <b>D.</b> Bar graphs show the size of viable tumor spheres after each treatment. <b>E.</b> Histogram shows cell cycle phase of treated tumor spheres. Data are shown as means ± SD (n = 5). *<i>P</i> < 0.01.</p

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 - Fig 4

<p><b>A.</b> Western blots show the expressions of p53, p21, E2F, and pAkt after OBP-301 or CDDP treatment. <b>B.</b> The scheme of cell cycle change.</p

Cell cycle distribution of MKN-45 cells forming tumors on Gelfoam^®.

<p>Sterile Gelfoam^® sponges (Pharmacia & Upjohn, Kalamazoo, MI), prepared from porcine skin, were cut into 1 cm cubes. The Gelfoam^® cubes were incubated at 37℃ in order that Gelfoam^® absorbed the RPMI 1640 medium. The absorbed Gelfoam^® was placed on agorose in 35 mm dishes. FUCCI-expressing MKN-45 cells were seeded on the absorbed Gelfoam^® for 3D culture. MKN-45 cells formed tumor-like structures on Gelfoam^® and spheres on the agarose. <b>A.</b> Schema of Gelfoam^® cultures on agarose. <b>B.</b> Macroscopic appearance of MRN-45 cancer cells forming tumor-like structures on Gelfoam^®. Images were acquired with the OV100 variable magnification fluorescence imager (Olympus, Japan). <b>C.</b> Macroscopic appearance of MKN-45 tumor-like structures growing on Gelfoam^®. Images were acquired with the OV100. <b>D.</b> Representative images of the MKN-45 tumor-like structure on Gelfoam^® using the FV1000 confocal laser scanning microscope (Olympus). <b>E.</b> Macroscopic images of MKN-45 spheres growing in agar. <b>F.</b> Microscopic FUCCI image of MKN-45 spheres.</p

The growing sciatic nerve intermingled with the dorsal root ganglion in 3D Gelfoam® histoculture.

<p>(A) A sciatic nerve was placed in Gelfoam® histoculture next to a dorsal root ganglion, both from a ND-GFP mouse. At day 38, many ND-GFP-expressing fibers were seen extending from both the sciatic nerve and co-cultured dorsal root ganglion. Bar: 500 µm. (B) Immunofluorescence staining of β-III tubulin (red) demonstrated that many β-III tubulin-positive fibers extended from both the sciatic nerve and the dorsal root ganglions. The fibers consisted of ND-GFP expressing cells. Bar: 500 µm. (C) Magnified image of the area inside the box in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067153#pone-0067153-g007" target="_blank">Figure 7B</a> shows that many β-III tubulin-positive fibers extended widely and radially both from the sciatic nerve and the dorsal root ganglion. β-III tubulin-positive fibers from both nerves intermingled with each other. Bar: 500 µm.</p

Location and characteristics of ND-GFP-expressing cells in the sciatic nerve.

<p>(A) The first day of Gelfoam® histoculture of a sciatic nerve bundle removed from the ND-GFP transgenic mouse. The sciatic nerve contained ND-GFP-expressing cells which had long processes (white arrows). Bar: 100 µm. (B) High-magnification image of the sciatic nerve showed that ND-GFP expressing cells and their processes are located between Schwann cells. Bar: 10 µm. (C) Transverse sections of the sciatic nerve of the ND-GFP mouse. The ND-GFP-expressing cells did not express β-III tubulin and S100. Bar: 10 µm. (D) Transverse and longitudinal sections of the sciatic nerve of the ND-GFP mouse. The ND-GFP-expressing cells (white arrows) co-expressed p75^NTR. Bar: 10 µm.</p

Intermingling of growing sciatic nerve in 3D Gelfoam® histoculture.

<p>(A) A sciatic nerve from a ND-GFP transgenic mouse was placed on Gelfoam® next to the sciatic nerve from an RFP transgenic mouse. At day 14, the sciatic nerve from the ND-GFP mouse was enriched with ND-GFP-expressing cells and intermingled with to the RFP-expressing sciatic nerve. Bar: 500 µm. (B) Magnified images of the area inside the box in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067153#pone-0067153-g006" target="_blank">Figure 6A</a> show that the ND-GFP-expressing cells proliferated in fibers growing from the nerve extending toward the other sciatic nerve. At day 9, the thickest fibers appeared between both sciatic nerves. At day 14, the two nerves intermingling with each other. Bar: 100 µm. (C) Magnified images of the area inside the box in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067153#pone-0067153-g006" target="_blank">Figure 6B</a> show that the fibers consisted of ND-GFP-expressing spindle cells (white arrow heads) and ND-GFP-negative spherical cells (white arrows). The spherical cells formed a line between both sciatic nerves and ND-GFP-expressing spindle cells extended among the lines. Bar: 10 µm. (D) A section of the intermingling two nerves. High-magnification images show that ND-GFP-expressing fibers growing from the sciatic nerve from the ND-GFP mouse invaded deeply into the RFP-expressing sciatic nerve. Bar: 100 µm.</p

The injured sciatic nerve expressed ND-GFP in vivo.

<p>(A) 1: The left sciatic nerve (green arrow heads) was exposed between the short and long adductor muscles. 2: ND-GFP expression was not observed. 3: The sciatic nerve was severed (black arrows: the nerve stumps). 4: The transected nerve was rejoined with a 9-0 suture (blue arrow head). (B) At day 0, left plantar (red arrows) was paralyzed. At day 35, the left plantar could show digit flection. (C) At day 35, the injured nerves (white arrow heads) connected (blue arrow head: point of suture). Strong nestin expression was observed in the injured sciatic nerve. Note that the intact nerve (white arrows) did not express ND-GFP. (D) The injured sciatic nerve (white dashed line) expressed ND-GFP. Nestin expression was stronger on the distal side compared to the proximal side of the sciatic nerve and spine (white arrow heads). White arrows: dorsal root ganglions.</p

Sphere formation and differentiation from the sciatic nerve in suspension culture.

<p>(A) The sciatic nerve was cultured in DMEM-F12 medium containing bFGF. The ND-GFP-expressing cells proliferated and formed spheres by day 32. Bar: 100 µm. (B) The spheres expressing ND-GFP co-expressed p75^NTR and CD34 but did not express β-III tubulin S100 and GFAP. Bar: 100 µm. (C) The ND-GFP-expressing spheres were switched to RPMI 1640 medium containing 10% FBS from DMEM-F12 containing B-27, N2 and bFGF and began to differentiate. At 7 days after switching into medium containing FBS, β-III tubulin-positive neuronal cells which expressed ND-GFP were observed. Bar: 10 µm. (D) At 14 days, the ND-GFP-expressing cells differentiated to GFAP-positive glial cells. Bar: 10 µm (E) At 7 days after culture in FBS medium, the ND-GFP-expressing cells differentiated to K15-positive keratinocytes, some of which still expressed nestin. Bar: 10 µm. (F) At 30 days after culture in FBS medium, the ND-GFP-expressing cells differentiated to α-SMA-positive smooth muscle cells. Bar: 50 µm.</p

ND-GFP-expressing cells proliferated in the sciatic nerve and formed fibers in 3D Gelfoam® histoculture.

<p>(A) Time-course imaging of ND-GFP-expressing cell trafficking in the histocultured sciatic nerve from an ND-GFP mouse. The nerve became enriched with ND-GFP-expressing cells by day 14. At day 6, the ND-GFP-expressing cells proliferated in extending fibers and migrated into the Gelfoam®. At day 14, many fibers consisting of ND-GFP-expressing cells extended widely and radially around the nerve. Bar: 500 µm. (B) Magnified images of the growing nerve. At day 6, the ND-GFP-expressing cells proliferated as fibers and migrated into the Gelfoam®. Bar: 100 µm. (C) High-magnification images of the area inside the boxes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067153#pone-0067153-g003" target="_blank">Figure 3B</a>. At day 9, the ND-GFP-expressing cells grew in fibers (white arrow heads). Some spherical cells proliferated in the growing fibers. They did not express nestin (white arrows). At day 14, the spindle cells expressed ND-GFP (white arrow head). In addition, there were spherical cells without nestin expression (white arrow). Bar: 10 µm.</p