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Responses of vascular endothelial cells to angiogenic signaling are important for tumor cell survival¹

SIQING SHAN^*, NICOLE D. ROBSON, YITING CAO^*, TONG QIAO, CHUAN Y. LI^*, CHRISTOPHER D. KONTOS, MARIANO GARCIA-BLANCO and MARK W. DEWHIRST^*,2

^* Department of Radiation Oncology,
Department of Medicine, and
Department of Genetics, Duke University Medical Center, Durham, North Carolina, USA

² Correspondence: Duke University Medical Center, Durham, NC 27710-3455, USA. E-mail: dewhirst{at}radonc.duke.edu

SPECIFIC AIMS

The objectives of this study were to investigate the effects of a soluble receptor of Tie-2, ExTek, and bFGF on murine mammary carcinoma 4T1 and melanoma B16—1) cell survival and proliferation after inoculation, 2) time of initiation of angiogenesis and 3) tumor growth, and 4) cellular expression of key angiogenic factors and receptors.

PRINCIPAL FINDINGS

1. Soluble Tie2 inhibited and bFGF promoted tumor cell adaptation, survival and proliferation in vivo. However, neither bFGF nor ExTek affected tumor cell proliferation in tissue culture
Dorsal skin fold window chambers were created in BALB/C or C57BL/6 mice and <100 4T1 or B16 cells, transfected with EGFP under control of a constitutive CMV promoter, were injected into the fascial of the chambers. Sustained release polymer pellets containing either 100 ng ExTek, 100 ng ExFms, or 50 ng bFGF were placed into the window chambers. Total cell numbers were counted from intravital fluorescence microscopy images taken on day 0, 2 and 4. 4T1 cell surviving fractions in ExTek- and ExFms-treated windows were reduced on day 2 (0.46 and 0.59, respectively). bFGF, however, protected tumor cells from death, as the relative cell number on day 2 was 0.92. Cell counts were higher in the bFGF group compared with ExTek and ExFms on day 2 (P<0.002 and 0.05, respectively). Tumor cell number was doubled in the bFGF group on day 4 (2.19 fold increase), which was greater than ExFms. Cell survival in ExTek-treated chambers was still lower than the original injected number on day 4 (0.89) (P<0.02 vs. bFGF) (Fig. 1 A). As shown in Fig. 1B , GFP-B16 cells displayed better survival than 4T1 with cell numbers being unchanged in control ExFms group on day 2. bFGF greatly promoted cell proliferation (1.64 fold on day 2 compared with that on day 0, P<0.02 vs. ExFms), while ExTek inhibited tumor cell survival (0.79 of day 0, P<0.05 vs. control). Most elongated 4T1cells polarized toward nearby blood vessels, and daughter cells showed the same orientation. The ratio of vessel-oriented 4T1cells on day 2 in the bFGF group was significantly higher than the ExTek group (P<0.02) (data not shown). In contrast to in vivo findings, cell growth of neither lines was affected in normoxic or hypoxic conditions in the presence of ExTek, ExFms or bFGF at concentrations equivalent to those used in vivo (Fig. 1C, D ).

View larger version (50K): [in this window] [in a new window]   Figure 1. A, B) 4T1 and B16 tumor cell survival and proliferation after inoculation. Columns represent median values for each group ( bFGF, ExFms, ExTek). Upper bars, 75 percentile; lower bars, 25 percentile. *, ** and *** denote P < 0.05, 0.02 and 0.002, respectively. Effects of bFGF and ExTek on proliferation of 4T1 (C) and GFP-B16 (D) cell lines in vitro. Tumor cells were seeded at 3 x 103/100 µl per well into 96-well plates in DMEM with 10% FBS and cultured overnight, then treated with one of three proteins ( bFGF, ExFms, ExTek), and a control without above proteins (). Each column represents the mean of 5 replicates (±SD). "N" and "H" indicate normoxic and hypoxic (O2 0.5%) conditions, respectively. E) RT-PCR of total cellular RNA of 4T1 and B16 cells. Shown are the RT-PCR products using PCR primers specific for FGFR1, FGFR2 FGFR3 and FGFR4. The arrow on the right indicates the expected uncut PCR product size. The far right panel represents restriction digest analysis of GFP-4T1 FGFR2 isotype expression. FGFR2 PCR products were digested with IIIb specific enzymes Hae III and Bsa I (lanes 2 and 3) and IIIc specific enzymes EcoRV and Hinc II (lanes 4 and 5). Uncut PCR is in lane 1. The arrow indicates the size of the uncut PCR product. F) Ang2, but not Ang1, is expressed in B16 tumor cell line and a murine endothelial cell line. RT-PCR was used to amplify a fragment of angiopoietins-1 and -2 and VEGF from different cell lines or plasmids. Cell lines included 4T1-GFP (4T1), B16 melanoma (B16), and Py-4-1 murine endothelial cells (Py). Plasmid cDNAs used as controls for specificity included murine Ang1 (A1+), murine Ang2 (A2+), or no template as a negative control (Neg). M, molecular weight markers.

2. Expression of angiogenic factors and receptors
Analysis of FGFR expression in both cell lines, using RT-PCR and restriction digestion with isotype specific enzymes, showed that most FGFR2 expressed by 4T1 is of the IIIb isotype which has low ligand affinity. B16 cells expressed only FGFR1, but not FGFR2 (Fig. 1E) . Angiopoietin (Ang) 1 was not expressed by either cell line, but Ang 2 was expressed in B16 cells only. Similarly, the cultured murine endothelial cell line Py-4-1 expressed Ang 2 but not Ang 1, while both tumor cell lines expressed VEGF (Fig. 1F) . Low levels of VEGF production were detectable in 4T1 and B16 cells under normoxia. VEGF was significantly up-regulated under hypoxic conditions, as measured by ELISA (data not shown).

3. Tumor growth and angiogenesis were significantly inhibited by ExTek and enhanced by bFGF
From day 6 through day 12, tumor size and VLD were measured every other day. In 4T1 tumors, the median area on day 8, day 10 and day 12 in ExTek-treated chambers was one third of the ExFms control group (P<0.05 on day 10 and 12) (Fig. 2 A). In contrast, bFGF significantly promoted tumor growth compared withExFms and ExTek. Total tumor area in bFGF chambers was larger than that in the ExFms group (P=0.05, P<0.05, P<0.02 on day 6, 8, and 12, respectively) (Fig. 2A ). In B16 tumors, a similar pattern was observed for these three proteins, but the differences were not statistically significant (Fig. 2B ). bFGF promoted early initiation of angiogenesis in 4T1 tumors (Fig. 2C) . Perfused blood vessels were readily observed in small tumor clusters on day 4, while no newly perfused blood vessels were observed in any tumor clusters in the other two groups. In contrast, ExTek significantly retarded the onset and progression of tumor angiogenesis. The differences in VLD among ExTek, ExFms and bFGF were highly significant from day 6 through day 12. Tumor VLD in the ExTek group remained significantly lower than the other two groups throughout the observation period. In B16 tumors, bFGF also promoted initiation of angiogenesis, although the ExTek did not show significant inhibition of tumor neovascularization when compared with controls (Fig. 2D ).

View larger version (50K): [in this window] [in a new window]   Figure 2. Effects of bFGF, ExTek and ExFms on tumor growth. Total areas of all tumor clusters were measured from images taken every other day from day 6 through day 12 (A for 4T1 tumor and B for B16). Tumor angiogenesis evaluated with vascular length density (VLD) in 4T1(C) and GFP-B16 (D) tumors. From day 4 (4T1) or day 6 (B16), VLD was calculated by dividing the total length of all vessels within tumor clusters or tumor mass and in areas closely adjacent to tumor cells. Columns represent median values for each group ( bFGF, ExFms, ExTek). Upper bars, 75 percentiles; lower bars, 25 percentiles. *, ** and *** denote P value less than 0.05, 0.02 and 0.002, respectively. E) Example showing ExTek inhibition on 4T1 tumor growth. The upper row, in an ExTek pellet-containing window. Lower row, a control window with ExFms pellet. The numbers of injected tumor cells were similar in both windows on day 0, however, the tumor cluster formation and tumor growth were significantly suppressed in ExTek window compared with ExFms on day 6 and day 12. Bars, 400 µm. F) 4T1 tumor regression in a window chamber treated with ExTek. Three tumor cell clusters are shown on day 6 (F1). They merged into a larger but less vascularized tumor mass (*) with a small cluster on day 8 to day 10 (F2 and F3). On day 12 (F4), the tumor mass regressed significantly leaving a small cluster of tumor cells. Bars: 200 µm in F1 and 500 µm in F2, F3, and F4.

CONCLUSION AND SIGNIFICANCE

This study demonstrated that Ang 2/Tie 2 and bFGF signaling are important not only for tumor angiogenesis but also for tumor cell survival prior to the onset of angiogenesis. Previously we reported that a soluble receptor of VEGF (ExFlk) significantly inhibited 4T1 cell survival and subsequent angiogenesis after inoculation into window tissue (Li et al., JNCI, 2000). These results suggest that tumor cell survival post-transplant may be dependent upon either paracrine signaling from the host vasculature or autocrine effects. Lack of any effect of ExFms, ExTek or bFGF on tumor cell proliferation in vitro suggests that autocrine effects are not likely. This point was further demonstrated by showing that the only FGF receptor expressed by the 4T1 tumor line has low affinity for bFGF, and B16 cells did not express FGFR2 at all. The chemotactic behavior of the 4T1 cell line toward host vessels provided further evidence for a paracrine communication pathway between tumor cells and the host microvessels. We showed B16 cells expressed Ang 2, and both tumor cell lines express VEGF, which has been shown to upregulate Ang 2 expression by endothelial cells. These cytokines may work in concert with bFGF to stimulate endothelial cells in existing host vessels. Aside from activating angiogenesis, this initial communication between tumor and endothelial cells may produce growth factors/cytokines that in turn affect tumor cell survival. Collectively, these results suggest that blockade of Ang 2/Tie 2, VEGF/Flk-1 and/or bFGF/FGFR2 signaling may inhibit early tumor cell survival prior to onset of angiogenesis. This opens the possibility of exploiting antiangiogenic therapies in the adjuvant setting, since such therapy might be effective in eliminating metastatic tumor cells even in sites where angiogenesis has yet to be initiated.

View larger version (58K): [in this window] [in a new window]   Figure 3. A hypothetical model of the mutual paracrine interaction between tumor cells and pre-existing host microvessels. Tumor cells produce angiogenic growth factors including VEGF, bFGF and angiopoietin 2 (B16 cells), which destabilize microvasculature and promote endothelial proliferation, migration, sprouting and tube formation via endothelial cell specific receptors. There is a syngeneic action between bFGF and VEGF, and VEGF induces Ang 2 upregulation of endothelial cells. It is possible that in response to these signals, endothelial cells up-regulate growth factor(s) or cytokine(s), which promote tumor cell survival and proliferation. Blockade of angiogenic signaling not only inhibits tumor cell induced angiogenesis, but also suppresses survival and proliferation of disseminated tumor cells at the pre-angiogenic stage.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.03-0765fje

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