The cervixes of estrogen-treated normal mice (N/E₂), HPV/E₂ mice with CIN3 lesions (at 3 mo of age), or with cervical squamous cell carcinomas, SCC (at 5 mo of age) were compared. Expression patterns were analyzed in at least five different mice of the same genotype or similar histological stage. Dotted line marks the epithelium-stroma boundary. E, epithelium; S, stroma; T, tumor.
(A) Immunostaining of PDGFR-α and -β (200× magnification; PDGFR, red; cell nuclei/DAPI, blue). Immunostaining of PDGF-CC (400× magnification).
(B) Coexpression of PDGF receptors and the mesenchymal cell marker vimentin in stromal fibroblasts of the transformation zone of HPV/E₂ mice (200× magnification; PDGF-α receptors, red; vimentin, green; merge; cell nuclei/DAPI, blue).
(C) Coexpression of PDGFR-β and the pericyte marker NG2 in the cervical stroma of the transformation zone of HPV/E₂ mice (400× magnification; PDGFR-β, red; NG2, green; cell nuclei/DAPI, blue).
(D and E) Quantitative PCR analysis of the expression of PDGF receptors (D) and ligands (E) in the cervixes of estrogen-treated normal or HPV mice with progressing tumor development. Error bars indicate the standard error of the mean.

(A) Representative immunostaining of blood vessels (CD31, green) in the cervical transformation zone of HPV/E₂ mice treated with vehicle or imatinib. Vessel density was analyzed in at least ten sections from five different mice of similar histological stage. Magnification is 200×; cell nuclei/DAPI, blue; dotted line marks the epithelium-stroma boundary. E, epithelium; S, stroma.
(B) Quantification of vessel density in the cervical transformation zone of HPV/E₂ mice treated in a prevention trial from 3.5 mo to 5 mo of age or in an intervention trial from 5 mo to 6 mo with vehicle or imatinib. Student t-test, t(CIN3) = 4.6, p < 0.001, t(SCC) = 3.8, p < 0.01.
(C) Visualization of pericytes and endothelial cells, using the markers NG2 (green) and CD31 (red), respectively. Pericyte coverage was analyzed in at least ten sections from five different mice of similar histological stage. Magnification is 200×; cell nuclei/DAPI, blue; dotted line marks the epithelium-stroma boundary; arrows indicate coexpression. E, epithelium; S, stroma.
(D) Quantification of pericyte coverage in the cervical transformation zone of HPV/E₂ mice following a Prevention Trial or an Intervention Trial. Student t-test, t(CIN3) = 4.5, p < 0.001, t(SCC) = 3.6, p < 0.01.
Error bars indicate the standard error of the mean.

(A) Analysis of cells isolated by FACS from the cervixes of 3.5-mo-old HPV/E₂ mice by sorting for expression of PDGFR-α and -β or c-kit. RT-PCR was performed to assess the expression of FGF-2, the squamous epithelial marker K14, the fibroblast cell marker vimentin, and the housekeeping gene L19 as a loading control.
(B) Representative immunohistochemical staining of FGF-2 in the transformation zone of the uterine cervixes of HPV/E₂ mice that had or had not been treated with imatinib displaying CIN3 lesions. The expression pattern was analyzed in at least five different mice of similar histological stage from each treatment group. Parameters: 400× magnification; dotted line marks epithelium-stroma boundary. As a control for specificity, the primary antibody was pre-blocked by incubation with recombinant mouse FGF-2 . E, epithelium; S, stroma.
(C) Expression of FGF-2 analyzed by quantitative RT-PCR following a long (14 d) and a short (3 d) treatment with imatinib (Student t-test, t = 3.5, p < 0.05). Expression of FGF-2 analyzed by quantitative RT-PCR following a 3-d treatment with control IgG or inhibitory antibodies against PDGFR-α and PDGFR-β (Student t-test, t = 3.1, p < 0.05). Note that for technical reasons, a different primer set was used in this experiment compared to the experiment shown in Figure 4C, yielding different absolute values of expression (for details, see Materials and Methods). Error bars indicate the standard error of the mean.

The transformed epithelial cells (purple) secrete VEGF, which becomes sequestered in the matrix until released by the action of MMP-9, produced by infiltrating macrophages (blue); the bioavailable VEGF then acts directly on endothelial cells (red) to stimulate angiogenesis. PDGF ligands are predominantly produced by the squamous epithelium and stimulate production of FGF-2 and FGF-7 by carcinoma-associated fibroblasts (brown) that express PDGF-receptors. FGF-2 stimulates angiogenesis by direct action on endothelial cells, and PDGF further promotes the angiogenic process by inducing pericyte (green) recruitment and association with newly formed blood vessels. FGF-7 may signal to the cervical carcinoma cells. Imatinib acts by inhibiting PDGF ligand-dependent PDGF receptor signaling (solid red lines), thereby repressing the production of FGF-2 and FGF-7 by carcinoma-associated fibroblasts (dotted red lines) and additionally reducing the pericyte coverage on tumor blood vessels. The clinically approved bisphosphonate zoledronate (zoledronic acid, Zometa, ZA) has been shown to suppress the expression of MMP-9 by macrophages (dotted red line), as well as to directly inhibit the proteolytic action of MMP-9 (solid red line).

(A) Tumor volume of carcinomas in the uterine cervixes of cohorts of mice treated (or not) with imatinib for 4 wk in an intervention trial, ending at 6 mo of age. Mann-Whitney U test, U = 5, p < 0.05.
(B and C) Incidence (χ² test, χ² = 5.1, p < 0.05) (B) and tumor volume (Mann-Whitney U test, U = 46, p < 0.05) (C) of invasive tumors in the uterine cervix following a 6-wk-long prevention trial ending at 5 mo of age. Mice without carcinomas had CIN2–3 lesions.
(D and E) Analysis of the proliferative (Student t-test, t(CIN3) = 2.3, p < 0.05; t(SCC) = 3.6, p < 0.01) (D) and apoptotic index (Student t-test, t(CIN3) = 3.1, p < 0.01; t(SCC) = 3.6, p < 0.01) (E) of CIN and SCC lesions in the cervixes of HPV/E₂ mice treated in the prevention trial with vehicle or imatinib. Error bars indicate the standard error of the mean.

(A) Quantitative RT-PCR analysis evaluating expression of a set of growth and/or angiogenic regulatory factors in the neoplastic cervixes of 4-mo-old HPV/E₂ mice treated with imatinib for 2 wk. The results are expressed as the ratio of expression (as percentage of the ribosomal protein gene L19) of imatinib-treated mice versus vehicle-treated mice. Bar colors indicate relative expression levels during the neoplastic progression (green = down-regulated expression compared with normal estrogen-treated cervix; red = up-regulated expression compared with normal estrogen-treated cervix; and white = unchanged expression compared with normal estrogen-treated cervix).
(B and C) Quantitative RT-PCR analysis of FGF-7 (Student t-test, t = 15.9, p < 0.001) (B) and FGF-2 (Student t-test, t = 13.7, p < 0.001) (C) expression in the cervixes of: estrogen-treated normal mice (N/E₂); HPV/E₂ mice with CIN3 lesions (3 mo) or SCC (5 mo) and HPV/E₂ mice treated from 3.5 mo to 4 mo of age with vehicle or imatinib.
(D) Western blot (WB) analysis of FGF-2 expression following immunoprecipitation (IP) of FGF-2 from tissue lysates of neoplastic cervixes of mice untreated or treated with imatinib for 2 wk. Two individual tissue lysates are shown for each treatment, and every lysate for each treatment group consisted of the combined cervixes from five mice. Omission of the immunoprecipitating antibody was used as a negative control (No Ab-lane), and 50 ng of recombinant mouse FGF-2 was used as a positive control. Densitometric quantification is shown normalized to lane 1.
(E) Immunostaining for the mitogenic signaling receptor for FGF-2, i.e., FGF receptor-1 (FGFR-1), in CIN3 lesions of the uterine cervix from HPV/E₂ mice. Expression of FGFR-1 (green) was predominantly detected in the stroma and colocalized with a marker for endothelial cells (red, CD31). The expression pattern was analyzed in at least five different mice of similar histological stage. Parameters: 200× magnification; cell nuclei/DAPI, blue; dotted line marks epithelium–stroma boundary. Similar results were seen in analysis of cervical carcinoma lesions (unpublished data). Note that the scattered punctate shapes distal from the vasculature are non–cell-associated debris derived from the secondary antibody, as revealed by evaluation at high magnification and analysis of tissue sections in which the primary antibody was omitted. E, epithelium; S, stroma.
(F) Quantification of vessel density in the cervical transformation zone of HPV/E₂ mice at 4 mo of age following a 2-wk treatment with imatinib or 2 wk after a single treatment with adenoviral delivery of FGF-trap or control GFP. Student t-test, t = 5.8, p < 0.001.
Error bars indicate the standard error of the mean.

Expression of PDGF receptors and FGF-2 was assessed in human hysterectomy samples and in human cervical SCC lesions by immunohistochemistry. Representative pictures from analysis of a total of three separate human normal cervixes and SCC lesions are shown. PDGF receptor-α was exclusively expressed by stromal cells underlying the epithelium, whereas PDGF receptor-β was expressed by stromal cells and pericytes. Similarly, FGF-2 was expressed by stromal cells. Although qualitative, the staining intensity of FGF-2 is clearly lower in all of the normal human samples than in comparable analyses of neoplastic human cervixes, and consistent with the results from the mouse (Figures 4C and 5B, and unpublished data), where FGF-2 is expressed in the normal cervical stroma at low levels and clearly up-regulated in the neoplastic cervix. As in the mouse model, PDGF-CC was predominantly expressed by the cervical epithelium. Parameters: 400× magnification; dotted line marks epithelium-stroma boundary. E, epithelium; S, stroma; T, tumor.