A selective small-molecule inhibitor of c-Met suppresses keloid fibroblast growth in vitro and in a mouse model

Keloids, tumor-like lesions that result from excessive scar formation, have no definitive treatment modality. Activation of c-mesenchymal-epithelial transition factor (c-Met) promotes cell proliferation and survival. Selective c-Met inhibitors, such as PHA-665752, may attenuate the activity of keloid fibroblasts and reduce keloid formation. Here, we aimed to evaluate the effect of PHA-665752, a second-generation selective small-molecule inhibitor of c-Met, on human keloid fibroblasts in vitro and in a mouse model. We performed in vitro cytotoxicity assays, scratch tests, western blotting, and immunofluorescence on human keloid fibroblasts. We also injected human fibroblasts into severe combined immunodeficient mice and measured the degree of nodule formation and skin histologic characteristics. We found that keloid fibroblast migration was inhibited by PHA-665752. Inhibitor treatment was also associated with lower expression of members of the hepatocyte growth factor/c-Met pathway, and lower fibroblast activity and collagen synthesis. In the in vivo experiments, PHA-665752—treated mice had lower nodule volumes and weights, accompanied by less inflammatory cell infiltration and collagen deposition, than those in control mice. These findings showed that although an in vivo model may not accurately represent the pathophysiology of human keloid development, PHA-665752 suppressed keloid fibroblast activity by inhibiting the c-Met—related tyrosine kinase pathway.


Discussion
We observed increased collagen synthesis and overexpression of c-Met in keloid specimens, consistent with the results of previous studies. Upon treatment with PHA-665752, we observed lower keloid fibroblast viability than that in untreated samples. PHA-665752 functions by inhibiting HGF-stimulated c-Met autophosphorylation and the constitutive phosphorylation of downstream mediators of the HGF/c-Met pathway in multiple tumor cell lines 14 . These results suggest that PHA-665752 is a potent inhibitor of c-Met activity in keloid scars and interferes with the downstream HGF/c-Met pathway 14 . scratch test demonstrated that PHA-665752 inhibited www.nature.com/scientificreports/ keloid fibroblast migration. Since fibroblast migration is known to be mediated by TGF-β activation, our findings are consistent with a previous study that suggested PHA-665752 has an inhibitory effect on TGF-β signaling 17 .
After we determined the inhibitory effects of PHA-665752 on fibroblast survival, proliferation, and migration, we examined its effects on the expression of proteins that are involved in the formation of keloid scars. TIMP2 is an endogenous inhibitor of MMPs. High levels of TIMP2 lead to accumulation of the extracellular matrix (ECM), whereas low levels of TIMP lead to matrix proteolysis We found higher levels of MMPs and a lower TIMP2-to-β-actin ratio in cells treated with PHA-665752 than those in control cells. Although MMPs can be elevated during the wound-healing process 18 , concurrent MMP elevation and TIMP suppression suggest that PHA-665752 enhances MMP expression, resulting in less ECM accumulation. Consistent with this interpretation, the c-Met inhibitor also suppressed collagen synthesis. www.nature.com/scientificreports/ Given that keloids occur exclusively in humans, animal models have been difficult to develop and are rarely used 19 . We used the keloid animal model that was introduced by Fujiwara et al., who implanted keloid fibroblasts into the dorsal skin of SCID mice 20 . We found that nodule size in both the PHA-665752 injection and DMSO groups decreased over time; nodule volume decreased more quickly in the inhibitor group. Although it is unclear why the keloid nodules decreased in size even in the untreated group, it might have been caused by a natural regression, as keloids occur exclusively in humans. This phenomenon was also observed in the study by Fujiwara et al., but it is not typically observed in human keloid tissues. Keloid regression contributes to the difficulty of producing animal keloid models and implies that the treatment response may differ from a potential clinical response. However, the weights of the harvested nodules differed between the treatment and control groups, which suggested that PHA-665752 antagonized keloid tissue formation or maintenance.
Our study has several limitations. First, the in vivo model does not accurately represent keloid pathogenesis. Second, although the site of origin may affect keloid tissue responses to PHA-665752, we did not analyze our results based on tissue origin. Finally, more keloid tissue samples would have contributed to a more generalizable, reliable result.

Cell viability analysis. Cell viability was measured using Cell
Immunofluorescence. All samples were fixed in 4% paraformaldehyde overnight, permeabilized with 100% methanol for 2 min, and blocked in 5% BSA for 1 h at room temperature. Samples were incubated with primary antibodies against collagen I (Col I, 1:1,000) and collagen III (Col III, 1:1,000) diluted in a blocking buffer overnight at 4 °C. The samples were then stained with secondary antibodies (Alexa Fluor Registered 594 goat anti-mouse IgG [1:200]  www.nature.com/scientificreports/ for 20 min. The samples were scanned using fluorescence inverted microscopy (Leica Microsystems, Wetzlar, Germany). Fluorescence intensity was measured using ImageJ 1.36b imaging software (National Institutes of Health, Bethesda, MD, USA).
Migration assay. Human keloid fibroblasts (2.0 × 10 4 ) were seeded in a 24-well plate. After the cells attached to the plate, we created a straight-line scratch in each well using a 1-ml pipette tip. The cells were then treated with PHA-665752 (0, 2, 4, and 8 μM) in DMEM containing 2% FBS and 1% A/A for 24 h. Images of the wound gap area at the initial time (0 h) and 24 h after the scratching were captured under an inverted fluorescence microscope (Leica Microsystems, Wetzlar, Germany). The area of the gap created by the scratch was calculated using ImageJ software and then compared to the size of the initial gap.
Experimental animals. The animal study was approved by the Institutional Animal Care and Use Committee of the Seoul National University Boramae Hospital (approval no. 2019-0031). Male NOD.CB17-Prkdc scid/J (SCID) mice (5 weeks old, body weight: 22.58 ± 0.91 g) were purchased from Charles River Japan (Yokohama, Japan). All animal experimental procedures were performed in accordance with ARRIVE guidelines 21  After washing three times with Tris-buffered saline containing 0.025% Triton X-100, the sections were incubated with secondary antibodies for 30 min at room temperature. Images (right, center, and left) were captured with an Olympus BX53 microscope. The optical density of each image was quantified using ImageJ 1.36b imaging software.
Antibodies. The following primary antibodies were used for western blotting, immunofluorescence, www.nature.com/scientificreports/ mined using the Mann-Whitney nonparametric U test, and multiple comparisons were tested with two-way analysis of variance. A p value less than 0.05 was considered as statistically significant. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.