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. 2009 Oct 13;106(41):17505-10.
doi: 10.1073/pnas.0908026106. Epub 2009 Sep 25.

Malignant cell-derived PlGF promotes normalization and remodeling of the tumor vasculature

Eva-Maria Hedlund  1 , Kayoko Hosaka, Zhaodong Zhong, Renhai Cao, Yihai Cao
Affiliations

Malignant cell-derived PlGF promotes normalization and remodeling of the tumor vasculature

Eva-Maria Hedlund et al. Proc Natl Acad Sci U S A. .

Abstract

Vascular functions of PlGF remain poorly understood and controversial. Here, we show that tumor cell-derived PlGF-1 and PlGF-2 displayed significant remodeling effects on the tumor vasculature, leading to a normalized vascular phenotype and improved functions against leakage. In two murine tumor models, that is, T241 fibrosarcoma and Lewis lung carcinoma, stable expression of PlGF-1 and PlGF-2 in tumor cells resulted in significant reduction of tumor microvascular density and branch formation. Markedly, the vasculature in PlGF-expressing tumors consisted of relatively large-diameter microvessels with substantial improvement of pericyte coverage. Similarly, PlGF-induced vascular normalization and remodeling were also observed in a spontaneous human choriocarcinoma that expressed endogenous PlGF. Our findings shed light on functions of PlGF as a vascular remodeling factor that normalizes the tumor vasculature and thus may have conceptual implications of cancer therapy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Comparison of tumor vasculatures induced by members in the VEGF family. (A) Vector-, VEGF-A165- and VEGF-C-expressing tumor tissues were immunostained using anti-CD31 (red) and tumor cells expressed enhanced green flurorescent (green). Arrowheads point to representative tumor microvessels. (Scale bar, 50 μm.) (B) PlGF-1- and PlGF-2-expressing tumors were immunostained using anti-CD31 (red) and tumor cells expressed enhanced green flurorescent (green). Arrowheads point to representative tumor microvessels. (Scale bar, 50 μm.) (C) Quantification of microvessel density in T241-vector, -VEGF-A, -VEGF-C, -PlGF-1 and PlGF-2 tumors using 20× magnification (n = 4–16 per group). (D) Quantification of microvessel density in T241-vector, -PlGF-1, -PlGF-2, and -VEGF-C tumors using 20× magnification (n = 4–16 per group), and the data were presented as mean (± SEM.).
Fig. 2.
Fig. 2.
Pericyte coverage in tumor vasculatures. (A–C) T241-vector, T241-PlGF-1, T241-PlGF-2, and T241-VEGF-A tumors were double immunostained using anti-CD31 (red) and anti-NG2 (green). Arrowheads indicate vasculature-associated pericytes. (Scale bar, 50 μm.) (D) Quantification of pericyte coverage in T241-vector, T241-PlGF-1, T241-PlGF-2, and T241-VEGF-A tumor vasculatures (n = 13–17 per group). All quantifications were performed using the 20× magnification. (E) Quantification of average diameters of tumor microvessels in T241-vector, T241-PlGF-1, T241-PlGF-2, and T241-VEGF-A groups (n = 6–10 per group). (F) Quantification of vascular branches of tumor microvessels in T241-vector, T241-PlGF-1, T241-PlGF-2, and T241-VEGF-A groups (n = 13–14 per group). All quantifications were performed using the 20× magnification, and the data were presented as mean (± SEM.).
Fig. 3.
Fig. 3.
Tumor vascular perfusion and leakiness. (A) T241-vector, T241-PlGF-1, T241-PlGF-2, and T241-VEGF-A tumor-bearing mice were intravenously injected with LRD (lysinated rhodamine-labeled dextran, 70 kDa, or 2,000 kDa). Extravasation of 70 kDa LRD (green fluorescence) was analyzed at 5 or 15 min respectively after injection. Tumor blood vessels were revealed by immunostaining using anti-CD31 (red). Arrowheads indicate extravasation of LRD from the tumor vasculature. Overlapping yellow indicates intravascular LRD. (Scale bar, 50 μm.) (B) Quantification of LRD extravasation in T241-vector, T241-PlGF-1, T241-PlGF-2, and T241-VEGF-A tumors using 20× magnification (n = 6–9 per group). (C) Quantification of 2,000 kDa LRD perfusion in T241-vector, T241-PlGF-1, T241-PlGF-2, and T241-VEGF-A tumors using 20× magnification (n = 4–6 per group), and the data were presented as mean (± SEM.).
Fig. 4.
Fig. 4.
PlGF-indcued vascular remodeling and normalization in LLC and human tumors. (A) T241-vector, T241-PlGF-1, T241-PlGF-2, T241-VEGF-A, LLC-vector, LLC-PlGF-1, LLC-PlGF-2, and LLC-VEGF-A tumors were double immunostained using anti-CD31 (red) and anti-NG2 (green). Arrowheads point to representative pericyte coverage in tumor vessels. (Scale bar, 50 μm.) (B) Human PlGF-expressing JE-3 choriocarcinoma and PlGF-negative A431 squamous carcinoma were double immunostained using anti-CD31 (red) and anti-NG2 (green). Arrowheads point to representative pericyte coverage in tumor microvessels. (Scale bar, 50 μm.) (C) Quantification of pericyte coverage in LLC-vector, LLC-PlGF-1, LLC-PlGF-2, and LLC-VEGF-A tumor vasculatures (n = 6–10 per group). (D) Quantification of microvessel branches in LLC-vector, LLC-PlGF-1, LLC-PlGF-2, and LLC-VEGF-A tumors (n = 7–10 per group). (E) Quantification of average diameters of tumor microvessels in LLC-vector, LLC-PlGF-1, LLC-PlGF-2, and LLC-VEGF-A groups (n = 6–10 per group). (F) Quantification of pericyte coverage of microvessels in JE-3 choriocarcinoma and A431 squamous carcinoma (n = 12–13 per group). All quantifications were performed using the 20× magnification, and the data were presented as mean (± SEM.).
Fig. 5.
Fig. 5.
Treatment of vector-T241 and PlGF-T241 tumors with VEGFR blockades. (A and D) PlGF-1-T241 tumors were treated with or without anti-VEGFR1 and pericyte coverage in tumor vessels were detected by anti-CD31 (red) and anti-NG2 (green). (B, C, E, and F) Vector tumors were treated with PBS, anti-VEGFR1 (MF1), anti-VEGFR2 (DC101), or sunitinib (sutent) and tumor vasculatures were detected using anti-CD31 (red) and NG2 staining (green). Arrowheads indicate vasculature-associated pericytes. (Scale bar, 50 μm.) (G) Quantification of vascular branches of tumor microvessels in various treated and non-treated groups. (n = 5–9 per group). (H) Tumor growth rates of T241-PlGF-1 fibrosarcoma treated with MF-1 (green line) or PBS (red line) (n = 6 mice per group). (I) Quantification of average diameters of tumor microvessels in various treated and non-treated groups (n = 5–11 per group). (J) Tumor sizes of T241-vector fibrosarcoma treated with MF-1 (green line), DC101 (blue line), or PBS (red line) (n = 6 mice per group). (K) Quantification of pericyte coverage in various vector and PlGF tumor vasculatures treated with various anti-VEGF agents (n = 5–10 per group). All quantifications were performed using the 20× magnification. (L) Tumor sizes of T241-vector fibrosarcoma treated with sunitinib (blue line) or PBS (red line) (n = 8 mice per group) and the data were presented as mean (± SEM.).
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