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. 2011 Dec;131(12):2467-76.
doi: 10.1038/jid.2011.300. Epub 2011 Sep 22.

Rapamycin suppresses self-renewal and vasculogenic potential of stem cells isolated from infantile hemangioma

Shoshana Greenberger  1 , Siming Yuan, Logan A Walsh, Elisa Boscolo, Kyu-Tae Kang, Benjamin Matthews, John B Mulliken, Joyce Bischoff
Affiliations

Rapamycin suppresses self-renewal and vasculogenic potential of stem cells isolated from infantile hemangioma

Shoshana Greenberger et al. J Invest Dermatol. 2011 Dec.

Abstract

Infantile hemangioma (IH) is a common childhood vascular tumor. Although benign, some hemangiomas cause deformation and destruction of features or endanger life. The current treatments, corticosteroid or propranolol, are administered for several months and can have adverse effects on the infant. We designed a high-throughput screen to identify the Food and Drug Administration-approved drugs that could be used to treat this tumor. Rapamycin, an mTOR (mammalian target of Rapamycin) inhibitor, was identified, based on its ability to inhibit proliferation of a hemangioma-derived stem cell population, human vasculogenic cells, which we had previously discovered. In vitro and in vivo studies show that Rapamycin reduces the self-renewal capacity of the hemangioma stem cells, diminishes differentiation potential, and inhibits the vasculogenic activity of these cells in vivo. Longitudinal in vivo imaging of blood flow through vessels formed with hemangioma stem cells shows that Rapamycin also leads to regression of hemangioma blood vessels, consistent with its known anti-angiogenic activity. Finally, we demonstrate that Rapamycin-induced loss of stemness can work in concert with corticosteroid, the current standard therapy for problematic hemangioma, to block hemangioma formation in vivo. Our studies reveal that Rapamycin targets the self-renewal and vascular differentiation potential in patient-derived hemangioma stem cells, and suggests a novel therapeutic strategy to prevent formation of this disfiguring and endangering childhood tumor.

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Conflict of interest statement

Conflict of Interest

The authors have no conflicts of interests to declare.

Figures

Figure 1
Figure 1. Rapamycin selectively inhibits the proliferation of HemSCs
(A) Dose-response curves of HemSCs (formula image),NHDF (formula image) and BM-MSCs (●) treated with Rapamycin. Error bars denote standard deviation (SD) (n = 32). (B) Quantification of PCNA-positive cells in HemSCs treated with Rapamycin. Error bars denote SD (n = 8). *P < .05 compared with the non-treated group. (C) Western blot for phospho4EBP-1(S65) and total 4EBP-1 in HemSCs serum-starved for 24 hours, pretreated with vehicle alone (“0”) or rapamycin for 20 minutes and then stimulated with media containing serum and growth factors for 30 minutes. (D) Bands in C quantified using ImageJ; normalized to β-actin. (E) Immuno-staining for phosphorylated AKT (green) and VE-cadherin (red), and staining for DAPI (blue), in proliferating or involuting IH. Scale bar, 50µm.
Figure 2
Figure 2. Rapamycin suppresses vessel formation in IH tumor model
(A) Dose-response to Rapamycin. Matrigel explants at Day 7. Scale bar, 1 cm. (B) MVD (erythrocyte-filled vessels). Mean value determined from explants ± the standard error of mean (SEM). N=6–8/group *P < .05 compared with vehicle-injected group. * *P < .05 compared with 0.1 mg/kg-injected group. (C) H&E of Matrigel explants from A. Scale bar, 100µm. (D) Immunostaining with anti-human CD31. Scale bar, 100µm. (E) MVD of Matrigel explants from mice injected systemically with Rapamycin, Everolimus or Temsirolimus. Bars and * as in (B). N=6/group. (F) Matrigel explants at Day 7; cells pre-treated with Rapamycin or DMSO. (G) MVD in Matrigel explants from mice injected with Rapamycin pre-treated HemSCs. Bars and * as in (B). N=7/group
Figure 3
Figure 3. Anti-vasculogenic effect of Rapamycin is beyond its anti-proliferative activity
(A) Cellular proliferation of HemSCs treated with increasing doses of Roscovitine. Bars denote the SD (n = 32). (B) Representative Matrigel explants at Day 7 containing cells pre-treated with Rapamycin, Roscovitine or DMSO as vehicle. Drugs were washed away from HemSCs before injection into mice. (C) MVD analysis of Matrigel explants in C. Bars denote mean value determined from all explants ± the SEM. N=8–10/group *P < .05 compared with the DMSO-pre-treated group. Experiment was repeated twice with similar results.
Figure 4
Figure 4. Rapamycin disrupts the stem-ness of HemSCs
(A) HemSCs pre-treated with Rapamycin or DMSO for 4 days; number of clones with > 10 cells after 9 days. (B) Percentage of clones (A) that expanded to indicated number of cells/well. (C) Quantitative PCR of LPL and C/EBP-α in HemSCs in adipogenic medium ± Rapamycin. (D) Oil Red-O staining of HemSCs in adipogenic medium ± Rapamycin. (E) Quantitative PCR of smMHC and SM22 in HemSCs ± Rapamycin. HemSCs co-cultured with cbEPCs, positive control. (F) Western blot for SM22 and αSMA in HemSCs ± Rapamycin. HemSCs co-cultured with cbEPCs, positive control. (G) Experimental design for H, I. (H) Matrigel explants at Day 7, described in G. (I) MVD of explants in H. Mean value ± SEM. N=7–8/group. *P < .05
Figure 5
Figure 5. Rapamycin stimulates regression of pre-existing vessels in IH tumor model
(A) Perfusion of microbubbles (green) into vehicle and Rapamycin injected mice at day 17, visualized with contrast ultrasonography. Matrigel borders are outlined in light blue. (B) Quantification of microbubble perfusion (contrast intensity above baseline) for individual mice injected with vehicle (red lines) or Rapamycin (grey lines). (C) Mean change of perfusion from day 7 to day 17 in vehicle or Rapamycin-injected mice. Error bars denote SEM. N=6/group *P < .05 compared with vehicle-injected group. (D) Matrigel explants at Day 17 (E) MVD analysis of Matrigel explants. Bars denote mean value determined from all explants ± the SEM. N=6/group. *P < .05 compared with the DMSO-pre-treated group. Experiment was repeated twice with similar results.
Figure 6
Figure 6. Rapamycin and corticosteroids target HemSCs by mutually exclusive mechanisms
(A) Quantitative ELISA analysis of VEGF-A protein in conditioned media of Rapamycin treated HemSCs. (B) Quantitative PCR analysis of the VEGF-A mRNA expression in HemSCs incubated with Rapamycin or dexamethasone (200 nM) (C) Protein array analysis 43 pro-angiogenic cytokines in conditioned media of HemSCs incubated with Rapamycin. (D) Representative Matrigel explants at Day 7 of mice systemically injected with dexamethasone, Rapamycin or the combination. (E) MVD analysis of Matrigel explants. Bars denote mean value determined from all explants ± the SEM. N=6/group *P < .05
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