Article Figures & Data
Figures
- Figure 1.
Milky spots are not necessary for tumor dissemination. Each experiment was done more than three times and showed similar results. A, representative en face findings of the toluidine blue-stained mouse omentum for detecting milky spot–like structures by a dissecting microscope. Note the cell aggregation on the narrow fat tissue stripe (right, arrowheads). B, representative en face findings of the toluidine blue-stained mouse mesentery for detecting milky spot–like and/or FALC structures by dissecting microscopy. No blue-stained cell aggregate can be seen in the mesentery from naïve mice (top). Bottom, a FALC-like structure in the mesentery from mice intraperitoneally treated with OK-432 (0.01K.E/head). Note the islands of cell aggregates (arrows). H&E, hematoxylin and eosin. C, representative en face findings of metastatic lesions on the mouse narrow fat tissue stripe 24 hours after intraperitoneal inoculation of CT26GFP (top). These disseminated lesions were completely diminished by simultaneous injection with AMD3100, a specific inhibitor for CXCR4. D, representative en face findings of metastatic lesions on the mouse mesentery 24 hours after intraperitoneal inoculation of CT26GFP (top). These disseminated lesions were also completely diminished by simultaneous injection with AMD3100. E, representative findings of the mouse omentum (top) and mesentery (bottom) 7 days after CT26GFP inoculation with or without AMD3100. F, graphs indicate the net weight of mouse narrow fat tissue stripes of the omentum (left graph) and mesentery (right graph). Note that AMD3100 almost abolished the weight gain due to the tumor dissemination.
- Figure 2.
Site-directed peritoneal dissemination targeting SCF+/CXCL12+ niche-like cells. Experiments were each done more than three times and showed similar results. CT26GFP cells (2 × 106) were injected intraperitoneally. A, grossly visible GFP+ peritoneal dissemination arises within 24 hours after intraperitoneal tumor cell injection. Note that a majority of disseminated tumor nodules were seen at the border regions of perivascular adipose tissue (inset A). Among the GFP+ nodules that were more than approximately 30 μm in diameter, 90.7% (mean) were located at the border area of perivascular adipose tissue (graph). B, protein expression level of CXCL12 at each site of the intraperitoneal tissue. The previously identified parietal peritoneum showed a lower level of CXCL12 than the mesentery or the narrow fat tissue of the omentum. C, representative en face findings of the mouse mesentery immunohistochemically stained for SCF (red) with CXCL12 (green), suggesting that some SCF+ cells would be CAR–like cells (white arrows). D, representative findings of the mouse mesentery on cross sections identifying the localization of CAR cells (CXCL12+/SCF+). The two top left panels show the tissue processing for perivascular adipose tissue (bracket, a branch of the mesenteric artery is indicated by elastica van Gieson staining) associated with the membranous region. The left bottom panel is a low powered view of the merged fluorescence (SCF, red; CXCL12, green; and nuclei, DAPI) and the hatched areas of A and B (in the membranous region), and that of C (at the border area of perivascular adipose tissue) correspond to the respective inset panels. Membranous regions were generally composed of SCF+/CXCL12− (inset B) and rarely showed SCF+/CXCL12+ CAR cells (inset A, arrow). In contrast, such doubly positive CAR cells were frequently seen at the border area of perivascular adipose tissue, as shown in inset C (white arrows). E, representative en face findings of the mouse mesentery immunohistochemically stained for SCF (red) as examined by a fluorescent microscope. Nuclear staining was done using DAPI (blue). Note the scattered solitary SCF+ cells showing typical reticular figures (white arrows). F, among the GFP+ nodules that were more than approximately 30 μm in diameter, 85.3% (mean) were on SCF+ cell sheets.
- Figure 3.
Facilitated spheroid formation of cancer cells in the peritoneal cavity is mediated by preexisting collagen type IV (Coll-IV) and plasma fibronectin (pFN). Each experiment was done more than three times and showed similar results. A, the representative time courses of the recovery of PCLF and mesenteric metastasis after intraperitoneal inoculation of CT26GFP cells. Single cells as well as scattered cell clusters were recovered without apparent metastasis within 1 and 3 hours after tumor inoculation. Grossly visible spheroids (arrowheads) as well as tumor nodules on the mesentery began to be observed from 6 hours after inoculation, and at 12 hours after inoculation, the size of spheroids was increased (arrows) and single cell recovery was very rare. B, PCLF facilitated sphere formation in vitro. Floating cultivation of CT26 was done in the fresh medium (fresh RPMI) as well as the medium that was used after intraperitoneal irrigation (PCLF-RPMI). C, cell cycle inhibition by aphidicolin or mitomycin C did not affect the formation of disseminated nodules. D, spontaneous spheroid formation and PCLF-mediated facilitation of spheroid formation were sensitive to collagenase. In both cases, CT26 cells could not form spheroids. E, Coll-IV and pFN cooperatively facilitated spheroid formation in vitro. Coll-IV, but neither Coll-I nor plasma/cellular fibronectins (pFN and cFN), facilitated the sphere formation (green squares), compared with baseline (blue squares). Importantly, the combination of Coll-IV and pFN showed the optimal effect (red squares). F, involvement of Coll-IV and pFN in the spheroid formation. E and F, measurement of Coll-IV and pFN in murine PCLF by ELISA (E), and immunohistochemical detection of Coll-IV and pFN in the CT26 spheroids (F) recovered from the peritoneal cavity.
- Figure 4.
Spheroid formation-induced CXCR4 expression is Sp1 transcription factor-dependent. Each experiment was done more than three times and showed similar results. A, CXCR4 played a significant role during disseminated nodule formation. Just before intraperitoneal injection of tumor cells, 1 × 106 CT26GFP cells were treated with neutralizing αCXCR4, αCXCR7, both, or αCXCL12 antibody. Pretreatment with AMD3100 was also included. B, method and detection of the upregulation of CXCR4 expression via spheroid formation in vitro. Twenty-four hours after floating culture, the spheroids were subjected to FACS analysis after propagation with EDTA (5 mol/L) or to immunohistochemistry. C, neither AMD3100 nor αCXCR4 abrogated CT26GFP sphere formation. The right graph indicates the diameters of the sphere. D and E, Sp1 was essential for the sphere formation–induced CXCR4 expression. Spheroid-induced upregulation of CXCR4 was abrogated by pretreatment with the Sp1-specific inhibitor mithramycin A in a dose-dependent manner (D) and with Sp1-specific siRNA (E). F, Sp1 was essential for the mesenteric metastasis of GFP+CT26 cells. Mesenteric metastasis of GFP+CT26 cells was significantly inhibited by the pretreatment with mithramycin A without any effect on spheroid formation in vivo.
- Figure 5.
Modulation of the CXCR4 axis achieves chemotherapeutic sensitization against a murine malignant ascites model. A, Coll-L inhibited spheroid formation of GFP+CT26 cells in vivo. Coll-L (75 U/mL) administered twice, at the time of tumor cell injection and 6 hours later, significantly inhibited the spheroidal formation as well as mesenteric metastasis in vivo. All mice that received higher doses (i.e., 150 and 300 U/mL) died due to lysis of the mesenteric tissue associated with massive intestinal necrosis within 24 hours. Each experiment was performed three to five times with similar results. B, experimental design. At the time of intraperitoneal inoculation of CT26 tumor cells, each chemical at each concentration or buffer was administered simultaneously. Six hours later, 5-FU (50 mg/kg) or buffer was administered, except in Group C, and thereafter 5-FU or buffer was administered weekly. C, sensitivity of CT26 tumor cells in a monolayer or sphere formation to 5-FU and L-OHP in vitro. Sphere formation made the cells more resistant to both antitumor agents compared with the cells in a monolayer. Each group contained three animals. D, time course of body weight (B.W.) and survival (+, dead animals over the 100 days after tumor inoculation;
, surviving animals over the same period).
Additional Files
Supplementary Data
- Supplementary Figures - Supplementary Figure S1-7
Volume 76, Issue 2
