The pro-inflammatory peptide LL-37 promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells - PubMed (original) (raw)

The pro-inflammatory peptide LL-37 promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells

Seth B Coffelt et al. Proc Natl Acad Sci U S A. 2009.

Abstract

Bone marrow-derived mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) have been shown to engraft into the stroma of several tumor types, where they contribute to tumor progression and metastasis. However, the chemotactic signals mediating MSC migration to tumors remain poorly understood. Previous studies have shown that LL-37 (leucine, leucine-37), the C-terminal peptide of human cationic antimicrobial protein 18, stimulates the migration of various cell types and is overexpressed in ovarian, breast, and lung cancers. Although there is evidence to support a pro-tumorigenic role for LL-37, the function of the peptide in tumors remains unclear. Here, we demonstrate that neutralization of LL-37 in vivo significantly reduces the engraftment of MSCs into ovarian tumor xenografts, resulting in inhibition of tumor growth as well as disruption of the fibrovascular network. Migration and invasion experiments conducted in vitro indicated that the LL-37-mediated migration of MSCs to tumors likely occurs through formyl peptide receptor like-1. To assess the response of MSCs to the LL-37-rich tumor microenvironment, conditioned medium from LL-37-treated MSCs was assessed and found to contain increased levels of several cytokines and pro-angiogenic factors compared with controls, including IL-1 receptor antagonist, IL-6, IL-10, CCL5, VEGF, and matrix metalloproteinase-2. Similarly, Matrigel mixed with LL-37, MSCs, or the combination of the two resulted in a significant number of vascular channels in nude mice. These data indicate that LL-37 facilitates ovarian tumor progression through recruitment of progenitor cell populations to serve as pro-angiogenic factor-expressing tumor stromal cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

LL-37 mediates MSC migration and invasion through a G protein-coupled receptor. (A) FPRL1 expression on 3 different donor pools of MSCs analyzed by flow cytometry. (B) Graphic representation of MSC migration stimulated as indicated in a modified Boyden chamber. EGF and PMA were used at 10 ng/mL. (C) MSC migration after pretreatment of cells with 100 ng/mL pertussis toxin (Ptx), or preincubation of LL-37 and EGF with an anti-LL-37 neutralizing antibody (αLL-37). (D) Invasion of MSCs through Matrigel-coated inserts following stimulation as indicated. (E) MSC invasion after pretreatment of cells with Ptx or preincubation of LL-37 and EGF with αLL-37 antibody. *, P < 0.05; **, P < 0.01. (F) Lysates from LL-37-treated MSCs analyzed for ERK phosphorylation by Western blot. MSCs in the far right lane were pretreated with Ptx for 1 h before stimulation with LL-37 for 10 min. M = molecular weight marker. (G) Quantification of Western blot band intensity by densitometry (n = 3), plotted as a bar graph.

Fig. 2.

Fig. 2.

Inhibition of LL-37 significantly reduces engraftment of MSCs into ovarian tumors. Human ovarian tumor xenografts were established i.p. in SCID mice. Mice were treated with IgG or anti-LL-37 antibodies (αLL-37) twice a week for 4 weeks. ffLUC-labeled MSCs were injected 4 times at weekly intervals 1 day after the first weekly injection of antibody, then visualized by bioluminescence in live mice. (A) Representative images of MSC engraftment into ovarian tumors 7 days after each injection of MSC. (B) Quantification of luminescence units emanating from tumor-engrafted MSCs. Values are mean ± SE. *P < 0.05. (C) Expression of LL-37 (red) in ovarian tumor sections of IgG- and αLL-37-treated mice. MSCs were identified using an anti-ffLUC antibody (green) and are indicated by white arrows. Nuclei were detected with DAPI. Sections are magnified ×100. (Scale bar, 100 μm.) (D) High-powered image (×400) of tumor section fluorescently labeled as described. (E) Sequential section of D immunohistochemically stained with anti-LL-37 antibodies followed by hematoxylin counterstain. (F) Example of LL-37-expressing MSCs in perivascular areas. (Scale bar, 50 μm, D–F.)

Fig. 3.

Fig. 3.

Growth of ovarian tumor xenografts is diminished by neutralization of LL-37. (A) Graphic representation of tumor weights from IgG- (n = 10) and αLL-37-treated (n = 9) animals obtained after surgical removal. Values are mean ± SE. **, P < 0.01. (B) Representative images of tumors stained for Ki-67 with hematoxylin counterstain. Arrows indicate mouse stroma in human xenograft tumors. (Scale bar, 50 μm.) (C) Graphic representation of the average number of Ki-67+ nuclei per high-powered field. Values are mean ± SE. **, P < 0.01. (D) Expression of Ki-67 and LL-37 in tumor necrotic region from an αLL-37-treated mouse. (Scale bar, 100 μm.)

Fig. 4.

Fig. 4.

MSCs secrete increased levels of angiogenic and inflammatory mediators after LL-37 stimulation. (A) The concentration of LL-37 in conditioned medium taken from unstimulated MSCs in culture. (B) Serum-starved MSCs were treated with 5 μg/mL LL-37 for 48 h, then conditioned medium was analyzed by Luminex-based cytokine arrays. Values are mean ± SE. *, P < 0.05, **, P < 0.01. (C) Analysis of MSC-conditioned medium after treatment for 48 h as indicated by gelatin zymography. The representative image depicts the electrophorectic pro-MMP-2 (72 kDa) and active MMP-2 (62 kDa). MMP-9 (92 kDa) was not undetectable. (D) Quantification of zymography by densitometry. Intensity of the lower band (active MMP-2, 62 kDa) is plotted as a bar graph (n = 3). (E) Conditioned medium from untreated and LL-37-treated MSCs was added to HUVECs, then seeded onto Matrigel. Fluorescently labeled cells were monitored were for formation of capillary-like tubules. Photographs are representative of HUVECs after 2 h incubation.

Fig. 5.

Fig. 5.

LL-37 enhances the pro-angiogenic activity of MSCs. (A) LL-37 or the combination of FGF and VEGFA was added to cold Matrigel with or without MSCs and injected into nude mice (n < 6). The absence of growth factors and cells served as negative control. After 7 to 10 days, Matrigel plugs were surgically removed, fixed, sectioned, and stained by H&E. Representative images of vascular channels are shown. (Scale bar, 100 μm.) (B) The average number of vascular channels in each plug section was determined by counting 3 high-powered fields of view then graphically represented. Values are mean ± SE. *, P < 0.05, **, P < 0.01, ***, P < 0.001. (C) Example of MSCs in perivascular areas identified by Ki-67 staining. (D) Fluorescently labeled, serum-starved MSCs were seeded onto Matrigel in the presence of 5 μg/mL LL-37 or 10 ng/mL FGF2 and allowed to incubate overnight. Formation of tubules, indicative of their pericyte-like differentiation, was captured by microscopy at ×200 the next day.

Fig. 6.

Fig. 6.

Schematic illustration of effects of LL-37 and MSCs on ovarian tumor progression (see Discussion).

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References

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