Tumor cell-derived PDGF-B potentiates mouse mesenchymal stem cells-pericytes transition and recruitment through an interaction with NRP-1

Abstract

Background: New blood vessel formation, or angiogenic switch, is an essential event in the development of solid tumors and their metastatic growth. Tumor blood vessel formation and remodeling is a complex and multi-step processes. The differentiation and recruitment of mural cells including vascular smooth muscle cells and pericytes are essential steps in tumor angiogenesis. However, the role of tumor cells in differentiation and recruitment of mural cells has not yet been fully elucidated. This study focuses on the role of human tumor cells in governing the differentiation of mouse mesenchymal stem cells (MSCs) to pericytes and their recruitment in the tumor angiogenesis process.

Results: We show that C3H/10T1/2 mouse embryonic mesenchymal stem cells, under the influence of different tumor cell-derived conditioned media, differentiate into mature pericytes. These differentiated pericytes, in turn, are recruited to bind with capillary-like networks formed by endothelial cells on the matrigel under in vitro conditions and recruited to bind with blood vessels on gel-foam under in vivo conditions. The degree of recruitment of pericytes into in vitro neo-angiogenesis is tumor cell phenotype specific. Interestingly, invasive cells recruit less pericytes as compared to non-invasive cells. We identified tumor cell-secreted platelet-derived growth factor-B (PDGF-B) as a crucial factor controlling the differentiation and recruitment processes through an interaction with neuropilin-1 (NRP-1) in mesenchymal stem cells.

Conclusion: These new insights into the roles of tumor cell-secreted PDGF-B-NRP-1 signaling in MSCs-fate determination may help to develop new antiangiogenic strategies to prevent the tumor growth and metastasis and result in more effective cancer therapies.

Figure 1

Molecular characterization and the effect of TCM on proliferation of 10T1/2 mesenchymal stem cells. A. Identification of mesenchymal properties of 10T1/2 cells by determining the expression of epithelial or mesenchymal marker proteins using Western blot analysis. B. A comparative study of α-smooth muscle actin (α-SMA) and desmin expression level in 10T1/2 cells and AOSMC by Western blot analysis using specific antibodies. The bar graphs represent the quantitative data (mean ± SEM) from three separate experiments. P-values were determined by unpaired student's t-test. **p < 0.005 vs 10T1/2 and ***p < 0.001 vs 10T1/2. C. Detection of α-SMA and calponin in 10T1/2 cells and AOSMC using immunofluorescence. D. Effects of different TCM on 10T1/2 cell proliferation. 1, regular media (RM), 2, MCF-7 cells derived conditioned media, 3, MDA-MB-231 cells derived conditioned media and 4, Mia-Paca-2 cells derived conditioned media. P-values were determined by unpaired students't-test. *p < 0.01 vs RM; #p < 0.05 vs MCF-7-CM.

Figure 2

Mesenchymal to pericyte transition (MPT) by different tumor cell-derived conditioned media (TCM). A-B. Representative images of MPT of 10T1/2 mesenchymal cells grown in regular media or different tumor cell-derived conditioned media for 24 h or 72 h. Cells are immuno-stained with pericyte-specific marker α-SMA, X200. C. Imuno-Western blots of α-SMA and desmin in 10T1/2 cells grown in different TCM for 24 h. The bar graphs represent the quantitative analysis from three different experiments. P-values were determined by unpaired student's-test. *p < 0.01 vs control (RM).

Figure 3

Tumor cell secreted PDGF-B is involved in MPT induced by tumor cells. A. Representative immuno-Western blot image illustrates the effect of PDGF neutralizing antibody on MCF-7-CM-induced α-SMA expression in 10T1/2 cells. B. Representative Giemsa stained Photographs show that mouse mesenchymal stem cells are converted into pericytes by PDGF recombinant protein after 72 h treatment. C-F. Representative immunofluorescence images exhibit MPT and α-SMA expression in PDGF-B exposed 10T1/2 cells. G. G. Schematic illustration of mesenchymal stem cell into pericyte transition.

Figure 4

NRP-1 is required for PDGF-B mediated induction of α-SMA and desmin expression. A. Representative immuno-Western blot shows the effect of MCF-7-CM on NRP-1 expression in 10T1/2 cells. 1, RM and 2, MCF-7-CM. *p < 0.001 vs RM. B. Detection of the effect of NRP-1 antibody on PDGF-B-induced Desmin and α-SMA expression in 10T1/2 cells. The bar graphs represent quantification of the Desmin and α-SMA of three different experiments, P-values were determined by unpaired students't-test. *p < 0.001 vs controls; **p < 0.001 vs PDGF treated samples. Ns, non-significant with controls.

Figure 5

Recruitment/attachment of vascular SMC and TCM-induced newly formed pericytes from 10T1/2 mesenchymal stem cells on HUVEC-generated capillary-like structures on Matrigel. A. The photograph showing the attachment of GFP (pZsGreen1C1, GFP-producing vector) -transfected AOSMC on the newly formed capillaries by HUVEC on 3 D matrigel. B. Differential attachment of Q-dot labeled newly formed pericytes from 10T1/2 cells on the HUVEC generated capillaries by RM or different tumor cells derived conditioned media on 3 D matrigel. C. Number of attached Q-dot-labeled newly created pericytes under different tumor cells-conditioned media on capillary-like structures formed by HUVEC on Matrigel. The data are representative mean ± SEM of ten different capillary-like structures. *p < 0.0001 vs RM; #p < 0.05 vs MDA-MB-CM or PaCa-CM. D. Upper panel shows the capillary formation in different treatment conditions, and lower panel shows the attachment of Q-dot labeled 10T1/2 cells on the newly formed capillaries by HUVEC in different treatment conditions on matrigel. RM, Regular Media, MCF-7-CM, MCF-7 cell-derived condition media and MCF-7-CM + PDGF ab¹, MCF-7-CM neutralized by PDGF antibody. Quantification of attachment of Q-dot-labeled cells using software provided by Nikon fluorescent photo microscope. The represents mean ± SEM of 10 capillaries of each experiment of three different experiments. P-values were determined by unpaired students't-test. *p < 0.001 vs RM; **p <0.001 vs MCF-7-CM.

Figure 6

TCM-induced tumor angiogenesis and recruitment/attachment of Q-dot-labeled newly formed pericytes on blood vessels formed on gel-foam inserted into mice. A. Representative images show the effect of MCF-7-CM on in vivo angiogenesis. Gelfoams, soaked with water (NC), regular media (RM) or tumor cell-derived media (TCM), were transplanted into the subcutis of mice and angiogenesis was viewed under the microscope. B. The photograph showing the recruitments of q-dot labeled pericytes on in vivo angiogenesis in three different conditions [Gelfoam soaked with water (NC), regular media (RM) and tumor-cell-derived condition media (TCM)]. The bar graph represents the number of Q-dot-labeled blood vessels per field in different treatment conditions. The data represent mean ± SME of three different experiments. *p of <0.0012 vs negative controls (NC); **p <0.0001 vs RM. C. Diagrammatic illustrations of signaling pathways involved in tumor cells-generated PDGF-B-induced differentiation of pericytes from mesenchymal stem cells. The studies demonstrate that PDGF-B is responsible for the differentiation of pericytes from mesenchymal stem cells through PDGF-B-NRP-1 signaling pathway.