Intraperitoneal Mesenchymal Cells Promote the Development of Peritoneal Metastasis Partly by Supporting Long Migration of Disseminated Tumor Cells

<div><p>The human peritoneal cavity contains a small number of free cells of mesenchymal cell lineage. Intraperitoneal mesenchymal cells (PMC) play supportive roles in metastasis formation on the peritoneum. In this study, we found that PMC, when co-cultuerd with human gastric cancer cells, MKN45, enhanced the proliferation of MKN45 when cultured at low, but not high, cellular density. Also, PMC suppressed apoptotic cell death of MKN45 only under low density culture conditions. Time-lapse videoanalysis clearly demonstrated that PMC randomly migrated more vigorously than did MKN45, and strongly enhanced the migration behavior of co-cultured MKN45. In fact, the majority of MKN45 migrated together in direct physical contact with PMC, and the sum of migration lengths from original position of co-cultured MKN45 for 48 hours was approximately 10 times longer than that of MKN45 cultured alone. Our data suggest that enhanced migration can increase the chance of direct contact or positional proximity among sparcely distributed MKN45, which may bring survival advantages to tumor cells. This may be one of the important mechanisms of peritoneal metastasis, since only a small number of tumor cells are considered to be disseminated in the early step of metastasis formation on the peritoneum.</p></div

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.

<p>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.</p

Peritoneal nodules developed in nude mouse after IP injection of MKN45 cells (1×10⁶) and PKH26-labelled MLCs (5×10⁵).

<p>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).</p

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.

<p>In F, MLCs (1<b>×</b>10⁵) were added on culture inserts within the same well. Data shown is representative of results from 3 different experiments.</p

Effect of PMC on MKN45 is not mediated by soluble factors.

<p>MKN45 (1x104) were cultured with or without PMC (1x104) in 6-well culture dishes. In some wells, PMC were separately cultured in inserts with 0.4-μm pores to inhibit direct cell-cell contact. Then the ratio of proliferation at 7 days and apoptosis at 2 days of MKN45 were examined as described in the legends of Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154542#pone.0154542.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154542#pone.0154542.g002" target="_blank">2</a>. Data show mean±SEM of 2 different experiments.</p

Cells recovered from a patient with peritoneal metastasis were cultured for 2 weeks and their phenotypes were examined by FACS.

<p>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.</p

Effect of PMC on apoptosis of MKN45.

<p>(A) MKN45 (1x104 or 1x105) was cultured with or without PMC (1x104) in 6-well dish for 48 hours. The cells were immunostained as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154542#pone.0154542.g001" target="_blank">Fig 1</a>, and the percentages of annexinV(+) 7AAD(+) cells in CD326(+) population were calculated. Data show mean±SD of 3 different experiments. (B) Representative FACS profiles of MKN45 (1x104) cultured with (lower panels) or without (upper panels) 1x104 PMC.</p

Summary of antibodies used in this study.

<p>Summary of antibodies used in this study.</p

Co-transfer of MLC enhances peritoneal metastasis of MKN45.

<p>MKN45 were intraperitoneally (IP) injected into 5 week Balb/c nude mice with or without indicated number of cultured MLCs The mice was sacrificed at 3 weeks later, and the number of mice which developed peritoneal metastasis was counted.</p

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.

<p>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.</p