Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Jul 21;23(14):2800-10.
doi: 10.1038/sj.emboj.7600289. Epub 2004 Jul 1.

VEGF-null cells require PDGFR alpha signaling-mediated stromal fibroblast recruitment for tumorigenesis

Jianying Dong  1 , Jeremy Grunstein, Max Tejada, Frank Peale, Gretchen Frantz, Wei-Ching Liang, Wei Bai, Lanlan Yu, Joe Kowalski, Xiaohuan Liang, Germaine Fuh, Hans-Peter Gerber, Napoleone Ferrara
Affiliations

VEGF-null cells require PDGFR alpha signaling-mediated stromal fibroblast recruitment for tumorigenesis

Jianying Dong et al. EMBO J. .

Abstract

We generated VEGF-null fibrosarcomas from VEGF-loxP mouse embryonic fibroblasts to investigate the mechanisms of tumor escape after VEGF inactivation. These cells were found to be tumorigenic and angiogenic in vivo in spite of the absence of tumor-derived VEGF. However, VEGF derived from host stroma was readily detected in the tumor mass and treatment with a newly developed anti-VEGF monoclonal antibody substantially inhibited tumor growth. The functional significance of stroma-derived VEGF indicates that the recruitment of stromal cells is critical for the angiogenic and tumorigenic properties of these cells. Here we identified PDGF AA as the major stromal fibroblast chemotactic factor produced by tumor cells, and demonstrated that disrupting the paracrine PDGFR alpha signaling between tumor cells and stromal fibroblasts by soluble PDGFR alpha-IgG significantly reduced tumor growth. Thus, PDGFR alpha signaling is required for the recruitment of VEGF-producing stromal fibroblasts for tumor angiogenesis and growth. Our findings highlight a novel aspect of PDGFR alpha signaling in tumorigenesis.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Generation of ras-transformed VEGF−/− MEF cell lines and fibrosarcomas. (A) A diagram of the strategy to derive ras-transformed VEGF−/− cells from the VEGF/loxp(+/+) mice, in which exon 3 of the VEGF gene is the target for deletion. (B) Quantitative RT–PCR analysis of VEGF RNA using primer/probe sets specific for exon 3, demonstrating the loss of VEGF exon 3 transcript in the VEGF−/− clones (G5, F10 and F4). (C) Fibrosarcoma formation by the parental VEGF+/+ C2P clone and the VEGF−/− clones. Tumor weight was determined 3 weeks after tumor cell implantation. (D) Sections of tumors originated from VEGF +/+ C2P and VEGF−/− G5 cells were stained for endothelial cell marker Flk-1. Vessel density in units/μm is indicated below the corresponding images.
Figure 2
Figure 2
Examination of VEGF RNA expression in tumors by in situ hybridization. Paraffin sections of tumors grown from VEGF+/+ C2P or VEGF−/− G5, F10 and F4 cell lines were hybridized with a 33P-labeled antisense riboprobe specific for the deleted exon 3 of VEGF sequence. (A–D) In C2P tumors, positive signal arises predominantly from tumor cells. The arrowheads indicate a continuous rim of hypoxic tumor and host stromal cells expressing increased levels of VEGF surrounding a necrotic area. (E–H) In G5 tumors, increased VEGF signal is noted in the hypoxic tumor zone (arrowheads); VEGF signal here is discontinuous, associated with stromal cells (arrows). (I–P) In F10 and F4 tumors, punctuate VEGF signal is noted at the boundary between necrotic tumor and viable tissue (arrowheads); the signal occurs in discrete regions consistent with origin in host stroma. Parallel images were taken with dark-field (A, C, E, G, I, K, M, O) or bright-field (B, D, F, H, J, L, N, P) illumination. Scale bars are 100μm (A, B, E, F, I, J, M, N) or 25μm (C, D, G, H, K, L, O, P).
Figure 3
Figure 3
Inhibition of C2P, G5 and F10 tumor growth by anti-VEGF treatment. Treatments were started 2 days post tumor cell inoculation by intraperitoneal administration of the anti-VEGF G6-23 or a control antibody anti-ragweed at 10 mg/kg, twice weekly. (A–C) Tumor growth was monitored by measurement with a vernier caliper. (D) Tumor weight was determined 3 weeks post tumor cell implantation. Statistical analyses were performed with Student's t-test comparing the anti-VEGF treatment groups with the control groups; *P<0.05.
Figure 4
Figure 4
Simulation of 3T3 fibroblast migration and proliferation by CM from ras-transformed MEF cells. (A) Relative fluorescence unit (RFU) indicates the relative number of chemotactic cells. (B) Proliferation activity was represented by [3H]thymidine incorporation as quantified by scintillation counting.
Figure 5
Figure 5
Partial purification of fibroblast chemotactic and mitogenic factors from G5 CM. (A) Fibroblast migration activity profile of fractions from the TSK size-exclusion column, which had been calibrated with known protein markers. The bioactive TSK fractions (28–31) were pooled and applied to the C4 Sepharose reversed-phase column. (B) Detection of PDGF AA in the active fractions from reversed-phase chromatography. The C4 column was eluted with a 15–50% gradient of acetonitrile. The collected fractions were tested for fibroblast proliferation activity and assayed for the presence of PDGF AA by ELISA.
Figure 6
Figure 6
Inhibition of G5 CM-induced fibroblast migration and proliferation by soluble PDGFR α-IgG. Migration (A) and proliferation (B) assays were conducted with G5 CM in the presence of different concentrations of either soluble PDGFR α-IgG or PDGFR β-IgG. Recombinant human PDGF AA and PDGF BB were included as controls.
Figure 7
Figure 7
Analysis of PDGF-A, PDGF-B, PDGF-C, PDGFR α and PDGFR β expression in G5 tumors by in situ hybridization. Paraffin sections of G5 tumors were hybridized with 33P-labeled riboprobes specific for PDGF-A, PDGF-B, PDGF-C, PDGFR α or PDGFR β as indicated. For each gene, antisense (columns 1, 3, 4) and control sense riboprobes (column 2) were applied to parallel sections. (A–D) PDGF-A expression is strong and uniform in the tumor mass. (E–H) PDGF-B expression occurs in discrete cell clusters consistent with vascular endothelial origin in tumors and is associated with vascular endothelial cells in the surrounding normal tissue (arrowheads at small arteriole in E, G, H). (I–L) PDFG-C signal is diffuse in tumors, and less strong than PDGF-A. (M–P) PDGFR α expression is associated with punctuate cell clusters consistent with stromal fibroblasts; no signal is associated with normal vessels in the surrounding tissue (arrowheads in M, O). (Q–T) PDGFR β expression is associated with stromal vessels (arrows in Q, S, T); positive signal is present in vascular smooth muscle in normal arterioles (arrowhead in Q). Parallel images were taken with bright-field (D, H, L, P, T) or dark-field (all others) illumination. Scale bars are 200 μm (A, B, E, F, I, J, M, N, Q, R) or 25 μm (C, D, G, H, K, L, O, P, S, T).
Figure 8
Figure 8
Inhibition of tumor growth by soluble PDGFR α-IgG and PDGFR β-IgG. (A) G5 tumor-bearing animals were treated with Av-LacZ, Av-PDGFR α-IgG or Av-PDGFR β-IgG once weekly. Tumor weight was determined 3 weeks later. (B) G5 cells in culture were infected with the indicated adenoviruses, and counted 5 days later. (C) ELISA was performed to measure VEGF protein concentrations in tumor lysates derived from different treatment groups. (D) C2P tumor-bearing animals were treated as described in (A). Student's t-test comparing the Av-PDGFR IgG treatment groups with the Av-LacZ group was performed to assess significance. P<0.05 was considered significant.
Figure 9
Figure 9
Multiple pathways involved in tumorigenesis by VEGF-null tumor cells. VEGF-null tumor cells secrete PDGF AA (and perhaps CC) to recruit host stromal fibroblasts through PDGFR α signaling pathway. The recruited host stromal fibroblasts in turn provide VEGF to simulate endothelial cell (EC) and to initiate angiogenesis. The stability of tumor vessels is dependent on the interaction between pericytes (PC) and endothelial cells (EC) via the PDGFR β signaling pathway.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abramsson A, Lindblom P, Betsholtz C (2003) Endothelial and nonendothelial sources of PDGF-B regulate pericyte recruitment and influence vascular pattern formation in tumors. J Clin Invest 112: 1142–1151 - PMC - PubMed
    1. Barbera-Guillem E, Nyhus JK, Wolford CC, Friece CR, Sampsel JW (2002) Vascular endothelial growth factor secretion by tumor-infiltrating macrophages essentially supports tumor angiogenesis, and IgG immune complexes potentiate the process. Cancer Res 62: 7042–7049 - PubMed
    1. Beckmann MP, Betsholtz C, Heldin CH, Westermark B, Di Marco E, Di Fiore PP, Robbins KC, Aaronson SA (1988) Comparison of biological properties and transforming potential of human PDGF-A and PDGF-B chains. Science 241: 1346–1349 - PubMed
    1. Bergers G, Song S, Meyer-Morse N, Bergsland E, Hanahan D (2003) Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors. J Clin Invest 111: 1287–1295 - PMC - PubMed
    1. Berking C, Takemoto R, Schaider H, Showe L, Satyamoorthy K, Robbins P, Herlyn M (2001) Transforming growth factor-beta1 increases survival of human melanoma through stroma remodeling. Cancer Res 61: 8306–8316 - PubMed

MeSH terms

Substances