Table 1.
Summary of antibodies used in this study.
Figure 1.
Intraperitoneal free cells were recovered from peritoneal lavages or ascites recovered at laparotomy from patients of gastrointestinal cancer and stained with FITC-conjugated anti-CD90, PE-conjugated anti-CD326 and PerCP-conjugated anti-CD45 and analyzed by FACS.
FACS profiles of a representative case were shown in A∼F. A: FSC/SCC, D: PerCP/PE, E: PerCP/FITC, F: FITC/PE. B and C show the FSC/SCC profiles of the cells located in Region 1 (CD45+, CD90−) and Region 2 (CD45−, CD90+), respectively. G: The cells were immunostained with FITC-conjugated anti-CD45 and PE-conjugated anti-CD90 mAbs and the ratio of CD45(−)CD90(+) cells were calculated in patients with or without peritoneal metastasis. The percentages of each cell population were calculated against total acquired cell counts of 104.
Figure 2.
Appearance of the peritoneal cells cultured for 7(A,D), 14 days (B,E) and 20 days (C,F).
A∼C: phase contrast, D∼F; photomicrograph following immunofluorescence staining. Cells were fixed and stained with FITC-conjugated anti-CD45 mAb and PE-conjugated anti-CD90 mAb, and two images to detect FITC and PE were merged in D∼F. Representative images are shown.
Figure 3.
Cells recovered from a patient with peritoneal metastasis were cultured for 2 weeks and their phenotypes were examined by FACS.
The cells were detached from culture plate and fixed and permeabilized using BD Cytofix/Cytoperm (Becton-Dickinson, San Jose, CA) before immunostaining. At this time point, some small hematopoietic cells contaminated the bulk culture, but were excluded by gating on FSC/SCC profile. Green lines denote the fluorescent profiles of the indicated antigens and filled lines correspond to negative controls. Each value represents the percentages of the cells with positive expression for indicated antigens in 104 of gated cells.
Figure 4.
Ascitic cells derived from a patient with peritoneal metastasis were cultured for 1(A) and immunostained with anti-FABP4 Ab (D).
The cells were cultured with osteogenic or chondrogenic media for 18(B) or Truisine Blue (C), respectively. Also, Osteocalcin (E) or Aggrecan (F) was immunostained using specific Abs followed by FITC conjugated secondary Abs.
Figure 5.
PBMC (1×106) derived from healthy volunteers were stained with CFSE and cultured on plastic (A) or anti-CD3 coated (B∼F) plates in the presence or absence of the indicated number of MLCs for 4 days, and CFSE fluorescence intensities in CD3(+) T cells were analyzed with FACS.
In F, MLCs (1×105) were added on culture inserts within the same well. Data shown is representative of results from 3 different experiments.
Figure 6.
Phase contrast images of ascitic cells derived from a patient with peritoneal metastasis were cultured with (B) or without (A) 10 ng/ml TGF-β for 24 hours.
C: Expression of Type I Collagen was quantitatively evaluated using FACS. MLCs treated with (Red line) or without (Green line) 10 ng/ml TGF-β for 48 hours were detached, fixed, permeabilized and stained with rabbit Ab to Type I collagen as described Material and Methods. Shaded profile shows the negative control. Same MLCs were cultured with (G,H,I) or without (D,E,F) 10 ng/ml TGF-β for 48 hours, fixed with paraformaldehyde and stained with mouse mAbs to Vimentin (D,G), α-SMA (E,H), and FAP-α (F,I). The slides were then incubated with PE-conjugated secondary Abs, and analyzed by fluorescence microscopy.
Table 2.
Co-transfer of MLC enhances peritoneal metastasis of MKN45.
Figure 7.
Peritoneal nodules developed in nude mouse after IP injection of MKN45 cells (1×106) and PKH26-labelled MLCs (5×105).
A: Nodules were excised and observed under fluorescence microscope. MLCs engrafted in metastatic nodules are highlight by arrow heads. Tissue sections of the peritoneal nodules were stained using the Masson-Trichrome method and observed under light (B) and fluorescence microscopy (C), and merged (D).
Figure 8.
A: MLCs (1×104) derived from ascites of a patient with peritoneal metastasis were cultured in the presence of the indicated concentrations of Imatinib or Dasatinib for 3 days. Cell proliferation was assessed by MTS assay. Data of a representative experiment from 3 different experiments are shown. B∼D: MKN45 cells (1×106) and MLCs (5×105) were co-injected into the peritoneum of nude mice. Dasatinib (50 mg/kg) in 1.0 ml PBS was orally administrated for 14 consecutive days starting 3 days after tumor inoculation. Two weeks later, the mice were sacrificed and macroscopic metastasis in the peritoneum were counted, excised and evaluated for the total weight (B). Tissue sections of the peritoneal nodules that developed in control (PBS) (C) and Dasatinib-treated (D) mice were stained using the Masson-Trichrome method.