Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal (original) (raw)
VEGF deprivation leads to selective obliteration of immature vessels in a xenografted glioma tumor. To enable control of the level of VEGF elaborated by tumor cells, a conditional VEGF expression system was created in which expression of VEGF by a C6 glioma is repressed when tetracycline is added to the drinking water of tumor-bearing nude mice. With the aid of this system, we have shown previously that overexpression of VEGF results in hypervascularization and that VEGF contributed by the endogenous gene was insufficient to sustain all vessels generated under conditions of VEGF overexpression. The latter is evident by the finding that shutting off VEGF production from the transgene resulted in detachment of a fraction of endothelial cells from the walls of some preformed vessels and their subsequent death by apoptosis. Vascular collapse led, in turn, to massive hemorrhages and extensive tumor necrosis (27). This system was used here to determine whether vulnerability to VEGF deprivation is associated with the state of vessel maturation.
Tumors were first allowed to grow for several weeks under conditions of maximal VEGF expression. After resection we determined the vessel maturation index, defined as the fraction of vessels that are associated with α-SMA–positive periendothelial cells. This measure was determined by scoring blood vessels larger than capillaries (i.e., vessels containing a lumen large enough for several erythrocytes), because capillaries are sparsely coated by pericytes and, therefore, may falsely appear as uncovered in thin sections. α-SMA–positive periendothelial cells could not be detected in the majority of these vessels, indicating that a large fraction of tumor blood vessels were immature (Fig. 1a). VEGF production from the transgene was then switched off in the remainder of the tumor-bearing animals. We have shown previously that this protocol leads to almost complete abrogation of transgene expression (27). Intermediate stages in vessel regression could be detected at 72 hours after switching off VEGF production and were evident by a loss in the continuity of the endothelial cell lining and by erythrocyte escape. Strikingly, evidence of vessel injury was only detected in vessels devoid of a periendothelial cell coating, including cases where α-SMA–negative vessels and α-SMA–positive vessels occupied the same tumor niche (Fig. 1, b and c). The outcome of this differential vulnerability was that by five days after VEGF withdrawal, the majority of the surviving vessels were found to have acquired an smooth muscle cell/pericyte coating (Fig. 1d; see also e for quantification of increase in the vessel maturation index). This result indicated that only immature tumor vessels depend on soluble VEGF for survival.
Regression of immature blood vessels in a xenografted glioma tumor. (a) A tumor grown under conditions of constitutive high-VEGF expression showing a mixture of α-SMA–positive and α-SMA–negative blood vessels. The α-SMA–negative vessel (arrow) is shown at a higher magnification in the inset (counterstained with H&E) to highlight the integrity of its endothelium. (b and c) A tumor 72 h after VEGF withdrawal. Both covered (right arrows) and uncovered (left arrows) vessels are still observed. However, the uncovered vessel shows clear evidence of disintegration (better seen in c depicting the same vessel in a serial section counterstained with H&E). (d) A tumor 5 days after VEGF withdrawal. Note that most surviving vessels are α-SMA–positive. (e) VMIs were determined, as described in Methods, in high-power fields of sections obtained either before or 5 days after VEGF withdrawal (scoring 270 or 87 vessels, respectively). Calculated VMIs were 0.30 (SEM = 0.04) and 0.94 (SEM = 0.05), respectively. H&E, hematoxylin and eosin; SMA, smooth muscle actin; VEGF, vascular endothelial growth factor; VMI, vessel maturation indices.
Established human tumors contain a significant fraction of vessels devoid of periendothelial cells. Before considering the targeting of immature tumor vessels it was essential to show that, like in the xenografted glioma models, natural human tumors contain a significant fraction of immature vessels. To determine the fraction of immature vessels present in a well-established glioblastoma, pathological specimens of glioblastoma multiforme were analyzed for the fraction of vessels that are α-SMA–positive. A representative field is shown in Fig. 2, depicting a high density of glomerulus-like vessels (a hallmark of glioblastoma multiforme) and smaller blood vessels nearby. Most of these blood vessels lack α-SMA–positive periendothelial cells. In the glioblastoma specimens examined, on average, less than one-quarter of tumor vessels larger than capillaries were associated with α-SMA–positive cells . The presence of a large fraction of immature vessels in glioblastoma multiforme was in sharp contrast to the normal brain where the majority of vessels were covered by α-SMA–positive cells (Fig. 2c). Glioblastoma multiforme is a fast-growing and highly angiogenic tumor. Therefore, it could be argued that the high proportion of immature vessels in this tumor reflects an exceptionally high rate of tumor growth and neovascularization and that slow-growing tumors may contain only an insignificant fraction of immature vessels.
Most blood vessels in glioblastoma multiforme are immature. (a and b) Serial sections of a glioblastoma multiforme tumor stained with anti-vWF (a) and with anti–α-SMA (b) showing only few α-SMA–positive vessels (arrows). (c) Four glioblastoma specimens were serially stained for α-vWF and α-SMA. Vessels larger than capillaries were scored (between 58 and 212 for each specimen), and the percentage of α-SMA–positive vessels is presented (averaging 19%; SEM = 8.3%). For comparison, vessels of a normal adult brain (rat) were also evaluated for the percentage of α-SMA–positive vessels (average of six high-power fields was 95%; SEM = 3%). vWF, von Willebrand factor.
To represent a slow-growing tumor, we analyzed prostate carcinoma specimens obtained from radical prostatectomies. Fields selected for vessel counting were centered around areas showing clear evidence of a carcinoma (as indicated by a specialized pathologist) and also contained normal and hyperplastic glands. On average, only ∼40% of vessels larger than capillaries were found to contain α-SMA–positive cells. Thus, even in regions containing much normal tissue, almost half of the blood vessels were immature. Interestingly, the healthy prostate gland also contained a significant fraction of immature vessels (see Fig. 4e).
Androgen-ablation therapy in prostate cancer leads to selective obliteration of immature vessels. Adjacent sections of surgically removed prostate tissues were immunostained for vWF (a and c) or for α-SMA (b and d) to examine individual vessels for coverage with periendothelial cells. One example is shown for an untreated tumor (a and b) (black arrows pointing at uncovered vessels and a blue arrow pointing at a covered vessel) and one for a tumor subjected to androgen-ablation therapy (c and d) (black arrows pointing at a covered vessel). Data from 10 different patients are shown in the histogram (e): five from control untreated tumors (hatched bars) and five from treated tumors (solid bars). To randomize for experimental variability during processing and immunohistochemical detection, pairs of tumors, each containing one control tumor and an androgen-ablated tumor of the same Gleason grade and a matching patient age, were embedded in a single block and coanalyzed on the same slide. The total number of lumenized vessels scored depended on the amount of tumor represented in the section and was as follows: tumor 1 (140), 2 (63), 3 (25), 4 (19), 5 (98), 6 (122), 7 (158), 8 (143), 9 (170), 10 (397). Mouse prostate was used as a control for vascular maturation in normal prostate. On average, untreated tumors contained 38% α-SMA–positive vessels (SEM = 3.5%); androgen-ablated tumors had 79% α-SMA–positive vessels (SEM = 3.3%).
Androgen-ablation therapy in prostate cancer leads to VEGF loss and selective obliteration of immature vessels. Recent studies in animal models showing that androgen deprivation may lead to vascular regression, in conjunction with findings that VEGF is androgen-regulated, prompted us to examine whether androgen-ablation therapy in human prostate carcinoma might induce the selective obliteration of immature vessels that depend on VEGF for survival.
To show that VEGF in the human prostate is indeed downregulated by androgen-ablation therapy, in situ hybridization analysis with a VEGF-specific probe was carried out. In untreated specimens VEGF was found to be abundantly expressed by the secretory epithelium of normal, hyperplastic, and cancerous glands. An example is shown in Fig. 3a; it depicts expression of VEGF by the secretory epithelium in normal glands and prostatic intraepithelial neoplasia (PIN). Androgen regulation of VEGF was demonstrated by comparing specimens of radical prostatectomy from untreated patients with those from patients subjected to androgen-ablation therapy for several weeks before surgery. In eight out of eight grade- and age-matched pairs cohybridized on the same slide, a dramatic reduction of VEGF expression was observed in the androgen-ablated specimens (for a representative example, compare Fig. 3, c and e with b and d). Hybridization of adjacent sections with another gene known to be downregulated in the prostate after androgen derivation, neutral endopeptidase 24.11 (29), showed that both genes are coexpressed in the same cells and are coordinately downregulated in the absence of androgen (data not shown). While a retrospective analysis of surgically removed human tumors is short of proving a causal relationship between androgen ablation and VEGF downregulation, a similarity to the situation in an animal model (26) is consistent with this proposition.
Downregulation of prostatic VEGF mRNA expression by androgen-ablation therapy. (a) In situ hybridization of a neoplastic prostate specimen with a VEGF-specific probe shown at high magnification. Note abundant expression of VEGF in the abnormal glandular epithelium. (b–e) Low-power magnifications to show global changes in VEGF expression via in situ hybridization of grade-matched specimens either untreated (b, bright-field; d, dark-field) or subjected to androgen-ablation treatment before prostatectomy (c, bright-field; e, dark-field). Sections were cohybridized on the same slide. Note a marked reduction in VEGF expression as a result of androgen-ablation therapy.
To determine whether VEGF loss has led to selective elimination of immature prostatic vessels, the fraction of vessels coated by α-SMA–positive cells was determined in untreated tumors and in tumors subjected to androgen-ablation therapy for several weeks before surgical removal. Shown in Fig. 4 are examples of the two types of vessels scored: vWF-positive/α-SMA–negative vessels (highlighted by black arrows in Fig. 4, a and b), and vessels of a comparable size that are vWF-positive/α-SMA–positive (highlighted by black arrows in Fig. 4, c and d). Quantification of each type of vessel in several high-power fields of each tumor specimen allowed the assignment of a vessel maturation index to each tumor. It was anticipated that a preferential vulnerability of immature vessels, and hence their selective obliteration, would result in an increase in the vessel maturation index. Indeed, untreated tumors contained only 38% coated vessels, whereas tumors resected 8–12 weeks after the onset of androgen-ablation therapy contained 79% coated vessels, suggesting that mature vessels are more refractory to this treatment (Fig. 4e).
In conjunction with an in situ apoptosis (TUNEL) analysis, these pathological specimens provided an opportunity to observe intermediate stages in vessel regression. In untreated tumors, TUNEL-positive endothelial cells were only rarely detected, and apoptotic cells were more often detected in the glandular epithelium (presumably reflecting a normal turnover of these cells) (Fig. 5a). In contrast, endothelial cell apoptosis was frequently detected after androgen ablation (Fig. 5b). Most informative were sections in which α-SMA–positive vessels were seen alongside α-SMA–negative vessels. Strikingly, in these cases endothelial cells undergoing apoptosis were predominantly found in α-SMA–negative vessels (Fig. 5c). These results provided conclusive evidence that a consequence of VEGF loss triggered by androgen deprivation is the selective obliteration of immature prostatic blood vessels.
Endothelial cell apoptosis after androgen ablation. TUNEL analysis was used to detect apoptotic cells in the untreated prostate and 4 weeks after hormone ablation. (a) An untreated prostate showing apoptotic nuclei (red) in glands but not in blood vessels (arrows). (b) An androgen-ablated specimen highlighting two blood vessels with several TUNEL-positive endothelial cells. Note the presence of TUNEL-positive (black arrow) and TUNEL negative (red arrow) in the same blood vessel. a and b were processed together on the same microscope slide to control for histochemical variability. (c) TUNEL (green fluorescence) and α-SMA staining (red fluorescence) showing that an uncovered blood vessel (arrowhead) contains many apoptotic endothelial cells, whereas an adjacent covered blood vessel (arrow) does not. Red autofluorescence of erythrocytes aids in identifying the lumen of these vessels. Eighty-five percent of vessels in which one or more TUNEL-positive endothelial cells were detected were α-SMA–negative. The mean number of TUNEL-positive endothelial cells per vessel was 3.1-fold greater in androgen-ablated tumors. TUNEL, terminal deoxynucleotide transferase–mediated dUTP nick end-labeling.