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Published ahead of print on September 8, 2005, doi:10.1165/rcmb.2005-0092OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 33, pp. 629-635, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2005-0092OC

Gene Transfer of the Vascular Endothelial Growth Factor Receptor flt-1 Suppresses Pulmonary Metastasis Associated with Lung Growth

Masahiro Mae, Timothy P. O'Connor and Ronald G. Crystal

Department of Genetic Medicine, and Division of Pulmonary and Critical Care Medicine, Weill Medical College of Cornell University, New York, New York

Correspondence and requests for reprints should be addressed to Ronald G. Crystal, M.D., Department of Genetic Medicine, Weill Medical College of Cornell University, 515 East 71st Street, Suite 1000, NY, NY 10021. E-mail: geneticmedicine{at}med.cornell.edu


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Growth of solid tumor metastases is critically dependent on angiogenesis. We hypothesized that an "angiogenic-rich" milieu, as in pneumonectomy-induced lung growth, would be conducive to growth of pulmonary metastases, and that transfer of an antiangiogenic gene would suppress tumor growth. Two weeks after left pneumonectomy in BALB/c mice, right lung mass increased 1.5-fold compared with controls (P < 0.0001). Our pulmonary metastases model, intravenous administration of {beta}-galactosidase ({beta}gal)–marked CT26.CL25 colon carcinoma cells, resulted in diffuse metastases at 12 d after administration. However, if left pneumonectomy was performed 1 d before tumor cell administration, right lung mass was increased 1.7-fold after 12 d (P < 0.001 compared with the right + left lung of controls), and {beta}gal activity was greater (2.8-fold, P < 0.05). To assess antiangiogenesis therapy, tumor cells were administered 1 d after pneumonectomy and 1 d later, 5 x 108 plaque-forming units of Adsflt (an Ad vector expressing the extracellular portion of the flt-1 vascular endothelial growth factor [VEGF] receptor) was administered. Compared with controls, mice receiving Adsflt via intranasal or intravenous routes showed suppression of pneumonectomy-induced tumor growth (P < 0.01, both routes compared with controls). Postpneumonectomy lung growth enhances growth of lung metastases, but this can be suppressed with Adsflt antiangiogenesis therapy.

Key Words: adenovirus-mediated gene therapy • antiangiogenesis • compensatory lung growth • lung metastasis • pneumonectomy


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The normal lung grows until the middle of the second decade in humans, when the fixed size of the chest cavity limits further growth despite the potential of lung parenchymal cells to proliferate and produce the extracellular components of the alveoli, airways, and blood vessels. However, if additional space is provided for lung growth, such as following pneumonectomy, and if the other thoracic structures are not fixed in place by mechanical constraints, there is increasing evidence that the adult lung is capable of compensatory growth (1). Although the evidence for postpneumonectomy lung growth in humans is necessarily indirect, this phenomenon is well documented in small mammals, in which unilateral pneumonectomy initiates a rapid, diffuse hyperplasia of the remaining lung cells, resulting in close to normal total mass, alveolar number, and function (2–9).

Whereas most attention to compensatory lung growth has been focused on the treatment of emphysema, there is the theoretical possibility that when pneumonectomy is used for removal of lung tumors, the availability of space signals the remaining lung to grow (2–8, 10–14). In this context, conventional therapy for many forms of tumors metastatic to the lung includes surgery to remove metastatic lesions identified by imaging and/or palpation at the time of surgery (15–17). Whereas the surgery is curative in some instances, recurrence of metastatic lesions within the lung are common (18). Although the signals controlling the growth of metastatic tumors are undoubtedly complex, there is indirect evidence that lung metastases may grow more rapidly after pneumonectomy (19–21).

Because the growth of new blood vessels is inherent to the growth of the lung, if the remaining lung does grow after pneumonectomy, the molecular signals involved must include mediators that induce angiogenesis. In fact, increased levels of angiogenic factors, such as insulin-like growth factor, hepatocyte growth factor, and erythropoietin, are found in models of lung growth, including postpneumonectomy compensatory growth, resulting in an "angiogenic milieu" (22–30). In the context of the growth of metastases, angiogenic mediators are particularly dangerous, because the process of neoangiogenesis within growing tumors uses similar mediators as does neoangiogenesis participating in the growth of normal tissue (13, 14, 31–33).

In the context of these considerations, we hypothesized that tumor cells metastatic to the lung will grow more rapidly in the milieu of postpneumonectomy lung growth, and that metastases growing in the lung parenchyma remaining after pneumonectomy must require proangiogenic signals for their growth. To assess these concepts, we have compared the growth of colon carcinoma metastatic to the lung in normal lung to that in the postpneumonectomy lung, and the ability of antiangiogenic therapy to suppress the growth of these tumors.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Cells
CT26 is an undifferentiated colon adenocarcinoma cell line (H-2 d) that was derived by administration of N-nitroso-N-methylurethane to a male BALB/c mouse. CT26.CL25 was derived from CT26 cells modified to express the Escherichia coli {beta}-galactosidase ({beta}gal) gene (kindly provided by N. P. Restifo, National Cancer Institute, Bethesda, MD) (34). This cell line was maintained in complete RPMI 1640 medium supplemented with 10% heat-inactivated FBS (Life Technologies, Grand Island, NY), 400 µg/ml G418 (Life Technologies), 2 mM glutamine, 100 µg/ml streptomycin sulfate, and 100 U/ml penicillin G.

Adenovirus Vectors
The replication-deficient adenovirus vectors used in this study are based on human adenovirus type 5 genome with E1 and E3 deletions and the expression cassette in the E1 region. Adsflt expresses a naturally occurring, soluble, secreted form of the human flt-1 receptor, under control of the constitutive cytomegalovirus early/immediate promotor/enhancer (35). AdNull has no transgene, and is used as a control (36). Both Ad vectors were propagated in 293 cells, purified by two rounds of cesium chloride density gradient centrifugation, dialyzed, titered by plaque-forming assay on 293 cells, and demonstrated to be free of replication-competent adenoviruses (37–39).

Experimental Model
Male BALB/c (H-2 d) mice, 6–8 wk old, were obtained from Taconic (Germantown, NY). Animals were housed under specific pathogen-free conditions and treated according to National Institutes of Health Guidelines.

To carry out unilatereral left pneumonectomy, mice were anesthetized with a combination of ketamine (100 mg/kg) and xylazine (10 mg/kg) by intraperitoneal injection. The trachea was cannulated with a 22-gauge angiocatheter (Becton Dickenson, Franklin Lakes, NJ) and mechanical ventilation was achieved via a small animal ventilator (Harvard Apparatus Inc., Holliston, MA). After a left thoracotomy in the third intercostal space, the left mainstem bronchus was carefully isolated and tied at the hilum with silk suture, followed by cutting distal to the ligation for removal of the left lung. The chest and skin were closed and mechanical ventilation was discontinued as spontaneous respiration was observed, and the animals were extubated. Mice in the sham groups underwent left thoracotomy in the same fashion, and the chest was closed without pneumonectomy. At the appropriate time points, mice were anesthetized and killed with a lethal dose of ketamine/xylazine, and the right lungs were removed and assessed as described subsequently here.

A metastatic lung tumor model was established by injecting 3 x 105 CT26.CL25 tumor cells in 100 µl of PBS (pH 7.4) via the jugular vein of syngeneic BALB/c mice. To characterize the time course of lung metastasis growth to optimize the Adsflt treatment and evaluation schedule, mice were killed 6, 9, 12, and 14 d after tumor injection. At each time point, lungs of one group (n = 5) were harvested, and lung wet and dry weight was measured as described subsequently here. In the other group (n = 5), lungs were weighed and {beta}gal activity of the lungs was quantified.

To assess the effect of compensatory lung growth after pneumonectomy on the growth of lung metastases, the 3 x 105 CT26.CL25 tumor cells in 100 µl of PBS were administered via the jugular vein of syngeneic BALB/c mice. Mice received left pneumonectomy or sham operation 14, 7, 4, or 1 d before tumor injection or 4, 2, or 1 d after tumor injection (5 mice per group for each time point). Twelve days after tumor injection, lungs were harvested, weighed, homogenized with lysis solution, and {beta}gal activity was measured and adjusted for total protein.

Quantification of Lung Growth with and without Metastases
Overall lung growth with and without metastases was determined by quantification of lung weight (right lung in the pneumonectomy group; right and left lungs separately in the sham group) and by quantification of lung {beta}gal activity (35, 40). Lungs were harvested en bloc and dissected away from the heart and thymus. The lungs were immediately weighed (wet weight) and then placed in a desiccating oven at 65°C for 48 h to measure dry weight. The ratio of wet/dry weight was used to quantify lung water content. The {beta}gal reporter gene in the CT26.CL25 colon carcinoma cells was assessed in lung lysates in a luminometer using Galacto-Light Plus kit (Tropic Inc., Bedford, MA). {beta}gal activity in the lung was expressed as total activity/lung and relative to total lung protein determined using the BCA assay (Bio-Rad Laboratories, Hercules, CA).

Histology
To assess the morphology of the lung, the lungs were fixed with 4% paraformaldehyde and embedded in paraffin. Butterfly-shaped sections of 5 µm thickness were cut and stained with hematoxylin-eosin (H&E; American Histoserve, Gaithersburg, MD).

Treatment of Lung Metastasis
To assess the effect of antiangogenic therapy with soluble flt-1 on the compensatory growth of the contralateral lung after pneumonectomy without tumor, Adsflt (5 x 108 plaque-forming units [pfu]) was administered intratracheally to mice after pneumonectomy; the same volume of PBS was administered as control (n = 5 mice). Lung wet weight was measured on Day 7. As a positive control for the Adsflt vector, studies were performed in mice with pre-established lung metastases as previously described (35). Metastatic lung tumors were established by injecting 3 x 105 CT26.CL25 tumor cells in 100 µl of PBS through the jugular vein of syngeneic BALB/c mice. The day after injecting tumor cells, mice were administered 5 x 108 pfu of AdNull in 50 µl of PBS through the intranasal route, or 5 x 108 pfu of Adsflt by the intravenous or intranasal routes (all groups, n = 5 mice). For intranasal administration, the mice were anesthetized, restrained, and placed on an operating surface at an angle of 60°. The Adsflt or AdNull vectors (5 x 108 pfu) resuspended in 50 µl of PBS were administered drop-wise into the nasal passages with an insulin syringe and 27-gauge needle over a period of 20–30 min per animal. Animals were killed 12 d after tumor cell injection. To quantify the tumor growth, lungs were weighed and {beta}gal activity of the lungs was measured (35, 40).

To assess the ability of Adsflt vector delivered to growing lung to suppress the accelerated lung metastatic tumor growth following unilateral pneumonectomy, 3 x 105 CT26.CL25 tumor cells in 100 µl of PBS were administered via the jugular vein of syngeneic BALB/c mice 1 d after left pneumonectomy or sham operation. One day after injecting tumor cells, mice were administered 5 x 108 pfu of Adsflt, AdNull, or no vector in 50 µl of PBS by the intranasal route, or 5 x 108 pfu of Adsflt in 100 µl of PBS by the intravenous route (5–6 mice per group). Animals were killed 12 d after tumor injection. To quantify the tumor growth, lungs were weighed, and {beta}gal activity of the lungs was measured as described previously here.

Statistical Analysis
All data are reported as mean ± SE. Statistical evaluation was performed with the two-tailed Student's t test. Correlations were assessed by linear regression analysis using StatView 5.0 (SAS Institute, Cary, NC).


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Compensatory Lung Growth after Left Pneumonectomy
Compensatory lung growth after left pneumonectomy in BALB/c mice was confirmed by measuring right lung wet weight. Postpneumonectomy right lung mass showed rapid growth, and increased by 30% at Day 5 (P < 0.0005), 45% at Day 7 (P < 0.0002), and 49% at Day 14 (P < 0.0001) compared with right lung wet weight in the sham surgery group (Figure 1). These observations demonstrate that left pneumonectomy leads to a rapid and complete compensatory lung growth response in the remaining lung, sufficient to restore normal mass by 14 d after surgery, with most of the lung growth occurring within the first 7 d. This compensatory lung growth was not suppressed significantly by intratracheal administration of Adsflt (lung wet weight in pneumonectomy with Adsflt was 127.9 ± 12.6 mg, compared with 130 ± 8.7 mg in pneumonectomy with PBS; P > 0.1).



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  		Figure 1. Compensatory lung growth after pneumonectomy. BALB/c mice underwent pneumonectomy of the left lung or sham operation. At the appropriate time points after surgery, the lungs were harvested and weighed. Zero time represents control animals (no surgery). Each data point represents mean ± SE (n = 5–6 animals per data point). Circles, right lung weight after pneumonectomy; squares, right + left lung weight after sham surgery; triangles, right lung weight after sham surgery.

 
Lung Metastases Model
In the context of lung metastases, postpneumonectomy lung growth was associated with enhanced tumor growth in the lung (Figure 2). The assessment was on Day 12 (rather than Day 14) because mice on Day 14 had increased respiratory rates and produced pleural effusion with hemorrhage due to extended lung metastases (data not shown). Lung histology of the mice with metastases showed an increased lung mass compared with the sham surgery animals, along with an increase in the mass of the tumor (Figure 2). The right and left lung histology in sham-operated mice 12 d after tumor injection showed micrometastases in perivascular regions and diffuse metastasis in subpleural regions (Figure 2A). In contrast, the right lung after pneumonectomy showed metastases greater in size and widely spread in the subpleural region (Figure 2B). Neither the sham lung nor the postpneumonectomy lung had significant inflammatory infiltration or lung edema, nor (in the animals with metastases) central necrosis in the tumors. Mice with lung metastases showed an increase in lung wet weight over time (Figure 3A). As a group, for control animals and those after pneumonectomy, comparison of lung wet weight to lung dry weight showed an excellent correlation (r2 = 0.99, P < 0.0001), demonstrating that changes in lung wet weight could be used as a valid indicator of increase of lung mass (e.g., the increase in wet weight was not secondary to edema; Figure 3B). In this context, the lung wet/dry weight ratio maintained the same level from Day 0 as control (4.7 ± 0.1) to Day 14 (5.1 ± 0.1; P < 0.001) after tumor injection. Assessment of the {beta}gal activity in the lungs of animals with metastases undergoing pneumonectomy demonstrated a good correlation of {beta}gal activity (i.e., volume of tumor) with lung wet weight (r2 = 0.85, P < 0.001) (i.e., measurement of {beta}gal activity and lung wet weight could be used interchangeably as a good indicator of tumor growth in the animals with lung metastases [Figure 3C]).



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  		Figure 2. Lung histology (hematoxylin and eosin staining) demonstrating enhanced right lung metastases after pneumonectomy compared with both lungs after sham operation. BALB/c mice underwent pneumonectomy or sham operation the day before (intravenous [IV]) administration of 3 x 105 CT26.CL25 tumor cells. Twelve days after tumor injection, the lungs were harvested and stained. (A) Right and left lungs after sham operation; (B) right lung after left pneumonectomy; scale bar = 5 mm; and (C) high-power view of subpleural lung metastasis in postpneumonectomy lung seen in (B); scale bar = 120 µm.

 


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  		Figure 3. Quantification of lung metastasis after pneumonectomy. BALB/c mice received (intravenously) 3 x 105 CT26.CL25 tumor cells. At various time points after tumor injection, the lungs were removed and weighed (wet), and then placed in a desiccating oven at 65°C for 48 h, to achieve dry weight. Zero time represents control animals (no tumor). Each data point represents mean ± standard error (n = 5 mice per data point). (A) Lung wet weight over time after tumor injection in the sham surgery group. (B) Correlation between lung wet and dry lung weight after tumor injection (r2 = 0.99, P < 0.0001). (C) Correlation between lung, {beta}-galactosidase ({beta}gal) activity (per mg) protein and lung wet weight. At different time points after tumor injection, lungs were harvested and weighed, then homogenized with lysis buffer. {beta}gal activity (per mg protein) was quantified as described in METHODS. {beta}gal Activity in the lung was expressed as total activity (relative light units [RLU]) normalized to total protein (r2= 0.85, P < 0.001). Each data point represents an individual animal. The correlations in (B) and (C) confirm that lung wet weight and dry weight are both valid indicators of the magnitude of tumor growth in animals with metastasis.

 
Enhanced Lung Metastases Associated with Postpneumonectomy Lung Growth
Quantification of lung metastases during compensatory lung growth after left pneumonectomy showed that lung metastases in the postpneumonectomy right lung markedly increased compared with lungs of mice treated with sham thoracotomy (Figure 4). The right lung mass 12 d after tumor administration was increased 1.7-fold (Figure 4A, P < 0.001) compared with the right + left lung of mice treated with sham thoracotomy. Likewise, {beta}gal activity of the postpneumonectomy right lung in the same experiment revealed significant greater activity than in mice treated with sham thoracotomy (Figure 4B, 2.8-fold, P < 0.05). Although pneumonectomy 1 d before tumor administration showed the most enhancement of postpneumonectomy lung metastases, mice undergoing pneumonectomy 4 or 7 d before tumor administration also showed significantly greater wet mass in right lung compared with the right + left lung in sham thoracotomy treated mice (P < 0.01; Table 1). Likewise, {beta}gal activity in the right lung of mice that received a left pneumonectomy 4 d before tumor administration was significantly greater than in sham operated mice (P < 0.05; Table 1).



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  		Figure 4. Enhanced lung metastasis after compensatory lung growth after pneumonectomy. Diffuse lung metastases were established in BALB/c mice by intravenous administration of 3 x 105 CT26.CL25 syngeneic tumor cells. Mice received pneumonectomy or sham thoracotomy 1 d before tumor injection. Twelve days after tumor injection, lungs were harvested, weighed and homogenized with lysis solution. {beta}gal activity (RLU) was measured and adjusted for total protein. (A) Wet weight. Shown is right (R) lung wet weight after pneumonectomy (Post-pnx) compared with right and left (R+L) lung wet weight after sham surgery. (B) {beta}gal activity. Shown is right lung {beta}gal activity after pneumonectomy compared with right and left lung {beta}gal activity after sham surgery ([A] P < 0.001; [B] P < 0.05).

 

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  		TABLE 1. TIME DEPENDENCY OF METASTATIC TUMOR GROWTH AFTER LEFT PNEUMONECTOMY*

 
Inhibition of Lung Metastases by Adsflt during Postpneumonectomy Lung Growth
Pre-established lung metastases were markedly suppressed by administration of 5 x 108 pfu of Adsflt by the intranasal or intravenous routes as compared with no vector (PBS) and AdNull groups (Figure 5). The lung wet weights were markedly reduced in the Adsflt-treated mice compared with both control groups (intranasal, P < 0.005; intravenous, P < 0.0005). Likewise, {beta}gal activity was markedly suppressed (intranasal, P < 0.005; intravenous, P < 0.01). The most efficient time point of gene delivery was the day after tumor injection, as compared with 2, 4, and 6 d after tumor injection, which yielded more variable results. When Adsflt was delivered intranasally 2, 4, or 6 d after tumor injections, lung weight was somewhat reduced, but did not differ significantly from the PBS or AdNull control groups. When delivered intravenously 2 or 4 d after tumor injections, lung weight in the Adsflt group was 50.3% (P < 0.01) and 68.1% (P < 0.02), respectively, of lung weight in the PBS and AdNull control groups (which did not differ significantly; P > 0.5 in both cases). {beta}gal activity in mice that had been administered intranasal Adsflt 2 or 4 d after tumor injections was 37.3% (P < 0.05) and 48.9% (P < 0.05), respectively, of the values in the PBS and AdNull control groups (which did not differ significantly; P > 0.2 in both cases).



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  		Figure 5. Suppression of pre-established lung metastases by Adsflt. CT26.CL25 tumor cells (3 x 105) were administered intravenously 1 d before 5 x 108 pfu of Adsflt, AdNull, or PBS was administered via intranasal or intravenous routes. Twelve days after tumor injection, the lungs were harvested and evaluated for (A) lung weight and (B) {beta}gal activity.

 
Lung histology demonstrated enhanced lung metastases in mice administered PBS or AdNull intranasally after left pneumonectomy compared with sham animals (compare Figures 6B and 6C to Figure 6A). In contrast, when Adsflt was administered either by the intranasal or the intravenous route 1 d after pneumonectomy, there was a marked suppression in the mass of metastases (Figures 6D and 6E). The postpneumonectomy right lung treated with Adsflt by the intranasal route showed smaller scattered metastases with minor inflammation (Figure 6D). The right lung treated with Adsflt by the intravenous route showed marked inhibition of perivascular metastasis without inflammatory changes (Figure 6E).



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  		Figure 6. Lung histology demonstrating effects of administration of Adsflt on growth of lung metastasis during postpneumonectomy compensatory lung growth. CT26.CL25 tumor cells (3 x 105) were administered intravenously 1 d after left pneumonectomy or sham operation. One day later, 5 x 108 pfu of Adsflt, AdNull, or PBS was administered intranasally or intravenously. Twelve days after tumor injection, the lungs were harvested and stained. (A) Right and left lungs for sham surgery group (no vector). (B–E) Right lung after pneumonectomy. (B) Intranasal PBS. (C) Intranasal AdNull. (D) Intranasal Adsflt. (E) Intravenous Adsflt. Hematoxylin and eosin; scale bar = 5 mm.

 
Quantitative analysis of lung metastases during postpneumonectomy lung growth demonstrated a marked inhibition when Adsflt was administered by intranasal or intravenous routes (Figure 7). Postpneumonectomy lung growth enhanced lung metastases in mice that had been administered PBS or AdNull by the intranasal route as compared with the right + left lungs of sham-operated mice without any treatment (lung weight, P < 0.05; {beta}gal, P < 0.02). The postpneumonectomy right lung weight in mice treated with 5 x 108 pfu of Adsflt by the intranasal or intravenous routes showed a significant decrease in lung mass (P < 0.005, intranasal route; P < 0.0001, intravenous route), as compared with PBS and AdNull treatments (Figure 7A). Consistent with this observation, {beta}gal activity in the postpneumonectomy right lungs of mice treated with Adsflt by the intranasal or intravenous routes also showed significant suppression compared with PBS and AdNull treatments (Figure 7B; P < 0.01, both routes). The changes in lung weight (P > 0.05) or {beta}gal activity (P > 0.7) were similar with intranasal or intravenous treatment with Adsflt. There was a discrepancy between the measurement of lung weight and {beta}gal activity (i.e., the weight was not reduced as much as the {beta}gal activity after nasal administration of Adsflt; this may be secondary to mild inflammation after intranasal administration of the Ad vectors [Figure 6]).



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  		Figure 7. Quantification of effects of administration of Adsflt on lung metastases during postpneumonectomy (Post-pnx) compensatory lung growth. The experiment was the same as described in Figure 6. Twelve days after tumor injection, lungs were harvested, weighed, and {beta}gal activity was determined. The data represent mean ± SE (n = 5–6 mice per group). (A) Lung weight. (B) {beta}gal activity. For each panel, shown is: right lung (R sham), sham surgery; right and left lung (R+L sham), sham surgery; right lung, postpneumonectomy (R post-px), PBS, nasal; right lung, postpneumonectomy, AdNull, nasal; right lung, postpneumonectomy, Adsflt, nasal; and right lung postpneumonectomy Adsflt, IV.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
This study is based on the hypothesis that tumor cells metastatic to the lung will grow more rapidly in the milieu of postpneumonectomy lung growth. Further, because metastases growing in the lung parenchyma remaining after pneumonectomy must require proangiogenic signals for their growth, it is likely that the rapid growth of lung metastases can be suppressed in the postpneumonectomy growing lung by antiangiogenic therapy. The results support this hypothesis. First, the growth of lung metastases was enhanced in the remaining lung during postpneumonectomy lung growth. Second, in vivo administration of an adenovirus vector encoding sflt-1, a soluble form of the flt-1 kinase receptor for the angiogenic factor vascular endothelial growth factor (VEGF), was effective antiangiogenic therapy, as evidenced by marked suppression of the enhanced growth of lung metastases during the postpneumonectomy lung growth.

Compensatory Lung Growth
Compensatory lung growth after lobectomy or pneumonectomy has been reported in experimental animals and humans for over 100 yr (6, 41). Many factors, such as stretch, oxygen concentration, blood flow, growth factors, and hormones that might potentially stimulate, modify, and/or regulate this response, have been studied (1, 4, 6, 11, 12, 26, 27, 42–49). Recently, it has been noted that several zinc finger genes as transcriptional regulators and immediate-early genes, such as c-fos, junB, and erg-1, are expressed during the rapid phase of compensatory lung growth (22, 23, 30). Cellular proliferation and differentiation in the remaining lung after pneumonectomy should promote tumor growth (19). In mice, removal of the left lung, which constitutes ~ 34% of total lung tissue, initiates compensatory growth rapid enough to restore normal total lung mass within 7–14 d (4). Although the specific mechanisms of compensatory lung growth are complex and remain incompletely understood (1, 49), it is known that postpneumonectomy lung growth is associated with an increase in the number of blood vessels in the lung (27, 30, 48), and thus proangiogenic factors must play a central role in postpneumonectomy lung growth. Interestingly, however, there have been no studies demonstrating a critical role of VEGF in postpneumonectomy compensatory growth. Our results, which showed no effect of Adsflt administration on compensatory growth, are consistent with the notion that alternative angiogenic factors are more critical for lung growth after pneumonectomy. The time course of transgene expression after adenovirus administration via the respiratory or intravenous routes is limited by immunity (primarily cellular) against the adenovirus vector per se. Although the intranasal route yields much higher levels of the transgene in the lung, the vascularization mechanisms of tumor growth is likely different than that of the normal lung and/or the relative numbers of VEGF receptors on the relevant endothelium may be different.

Antiangiogenic Therapy
When tumor size exceeds 1–2 mm3, the tumor becomes critically dependent on angiogenesis to continue growing (31, 50). Although the process of tumor neovascularization promoted by secretion of a number of angiogenic mediators is controlled tightly by a balance of activating and inhibiting factors, VEGF plays a central role in tumor progression and metastasis (32, 33, 51, 52). Based on the critical dependence of many tumors on VEGF, the strategy using a soluble form of the flt-1 VEGF receptor is predicted to be applicable to the treatment of many solid tumors (34, 35, 53). Under conditions of excess soluble flt-1 protein, VEGF is prevented from binding to its cognate cell surface receptors (i.e., soluble flt-1 functions as an efficient, dominant negative inhibitor of angiogenesis in vivo [33, 54]). Although this function might be overwhelmed by an excess of VEGF produced by the tumor cells, continuous overexpression of antiangiogenic factors by the administration of Adsflt shifts the balance in favor of antiangiogenesis within the tumor and leads to a reduction of solid tumor, including pulmonary metastases. Importantly, treatment with Adsflt markedly suppressed both established pulmonary metastases and postpneumonectomy-enhanced tumor growth as well. This suggests that an antiangiogenesis strategy could be an effective component of postsurgery treatment (see subsequent discussion), but could also slow the growth rates of established tumors in the absence of surgical interventions.

Pulmonary Metastases after Pneumonectomy
Whereas most attention regarding compensatory lung growth has been focused on lung overexpansion associated with the long-term development of emphysema and deteriorating pulmonary function, the observations in the present study suggest the theoretical possibility that pulmonary resection for treatment of tumors may result in conditions that promote compensatory lung growth and are also permissive of accelerating the growth of residual tumor. Further, surgical treatment for primary lung cancer or metastatic lung tumor includes manipulation procedures that may be implicated in hematogenous micrometastasis to the remaining lung (55). Surgery for advanced non-small cell lung cancer, such as stage IIIA or metastatic lung tumors, is controversial but curative in some instances, and may be occasionally associated with long-term survival. However, in some patients, secondary metastases and/or recurrence within the remaining lung appear soon after the first pulmonary resection (17, 20, 56). Although the signals controlling the growth of metastatic tumors are undoubtedly complex, the data in the present study suggest indirect evidence that lung metastases may grow more rapidly during compensatory lung growth after pneumonectomy. In this context, an antiangiogenesis strategy for tumors in the remaining lung after lung surgery, such as Adsflt-mediated gene therapy, may be useful to prevent the regrowth of micrometastases. The data in the present study suggest the time point of administration of antiangiogenic agents is important, and it is likely that antiangiogenic therapy should occur near the time of surgery.


   Acknowledgments
 
The authors thank L. Landesberg, P. Leopold, R. Kaner, and R. Singh in our laboratory for helpful discussions and assistance, and N. Mohamed for help in preparing this manuscript.


   Footnotes
 
This work was supported in part by Will Rogers Memorial Fund, Los Angeles, CA, and GenVec, Inc., Gaithersburg, MD.

Originally Published in Press as DOI: 10.1165/rcmb.2005-0092OC on September 8, 2005

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form March 3, 2005

Accepted in final form August 26, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

  1. Hsia CC. Signals and mechanisms of compensatory lung growth. J Appl Physiol 2004;97:1992–1998.[Abstract/Free Full Text]
  2. Cowan MJ, Crystal RG. Lung growth after unilateral pneumonectomy: quantitation of collagen synthesis and content. Am Rev Respir Dis 1975;111:267–277.[Medline]
  3. Holmes C, Thurlbeck WM. Normal lung growth and response after pneumonectomy in rats at various ages. Am Rev Respir Dis 1979;120:1125–1136.[Medline]
  4. Cagle PT, Thurlbeck WM. Postpneumonectomy compensatory lung growth. Am Rev Respir Dis 1988;138:1314–1326.[Medline]
  5. Hsia CC, Herazo LF, Fryder-Doffey F, Weibel ER. Compensatory lung growth occurs in adult dogs after right pneumonectomy. J Clin Invest 1994;94:405–412.
  6. Rannels DE, Rannels SR. Compensatory growth. In: Crystal RG, West JB, Weibel ER, and Barnes PJ, editors. The Lung, 2nd ed. Philadelphia: Lippinott-Raven; 1997. pp. 1035–1046.
  7. Takeda S, Wu EY, Epstein RH, Estrera AS, Hsia CC. In vivo assessment of changes in air and tissue volumes after pneumonectomy. J Appl Physiol 1997;82:1340–1348.[Abstract/Free Full Text]
  8. Takeda SI, Ramanathan M, Estrera AS, Hsia CC. Postpneumonectomy alveolar growth does not normalize hemodynamic and mechanical function. J Appl Physiol 1999;87:491–497.[Abstract/Free Full Text]
  9. Brown LM, Rannels SR, Rannels DE. Implications of post-pneumonectomy compensatory lung growth in pulmonary physiology and disease. Respir Res 2001;2:340–347.[CrossRef][Medline]
  10. Brody JS, Burki R, Kaplan N. Deoxyribonucleic acid synthesis in lung cells during compensatory lung growth after pneumonectomy. Am Rev Respir Dis 1978;117:307–316.[Medline]
  11. Hsia CC, Johnson RL. Physiology and morphology of postpneumonectomy compensation. In: Crystal RG, West JB, Weibel ER, and Barnes PJ, editors. The Lung, 2nd ed. Philadelphia: Lippincott-Raven; 1997. pp. 1047–1059.
  12. Langston C, Sachdeva P, Cowan MJ, Haines J, Crystal RG, Thurlbeck WM. Alveolar multiplication in the contralateral lung after unilateral pneumonectomy in the rabbit. Am Rev Respir Dis 1977;115:7–13.[Medline]
  13. Lefrak SS, Yusen RD, Trulock EP, Pohl MS, Patterson A, Cooper JD. Recent advances in surgery for emphysema. Annu Rev Med 1997;48:387–398.[CrossRef][Medline]
  14. Gierada DS, Yusen RD, Villanueva IA, Pilgram TK, Slone RM, Lefrak SS, Cooper JD. Patient selection for lung volume reduction surgery: an objective model based on prior clinical decisions and quantitative CT analysis. Chest 2000;117:991–998.[Abstract/Free Full Text]
  15. Mountain CF, McMurtrey MJ, Hermes KE. Surgery for pulmonary metastasis: a 20-year experience. Ann Thorac Surg 1984;38:323–330.[Abstract]
  16. Rena O, Casadio C, Viano F, Cristofori R, Ruffini E, Filosso PL, Maggi G. Pulmonary resection for metastases from colorectal cancer: factors influencing prognosis. Twenty-year experience. Eur J Cardiothorac Surg 2002;21:906–912.[Abstract/Free Full Text]
  17. Jungraithmayr W, Hasse J, Olschewski M, Stoelben E. Indications and results of completion pneumonectomy. Eur J Cardiothorac Surg 2004;26:189–196.[Abstract/Free Full Text]
  18. Kandioler D, Kromer E, Tuchler H, End A, Muller MR, Wolner E, Eckersberger F. Long-term results after repeated surgical removal of pulmonary metastases. Ann Thorac Surg 1998;65:909–912.[Abstract/Free Full Text]
  19. Brown LM, Malkinson AM, Rannels DE, Rannels SR. Compensatory lung growth after partial pneumonectomy enhances lung tumorigenesis induced by 3-methylcholanthrene. Cancer Res 1999;59:5089–5092.[Abstract/Free Full Text]
  20. Maniwa Y, Okada M, Ishii N, Kiyooka K. Vascular endothelial growth factor increased by pulmonary surgery accelerates the growth of micrometastases in metastatic lung cancer. Chest 1998;114:1668–1675.[Abstract/Free Full Text]
  21. Brown LM, Welch DR, Rannels SR. B16F10 melanoma cell colonization of mouse lung is enhanced by partial pneumonectomy. Clin Exp Metastasis 2002;19:369–376.[CrossRef][Medline]
  22. Dovat S, Gilbert KA, Petrovic-Dovat L, Rannels DE. Targeted identification of zinc finger genes expressed in rat lungs. Am J Physiol 1998;275:L30–L37.
  23. Gilbert KA, Rannels DE. Increased lung inflation induces gene expression after pneumonectomy. Am J Physiol 1998;275:L21–L29.
  24. Nobuhara KK, Di Fiore JW, Ibla JC, Siddiqui AM, Ferretti ML, Fauza DO, Schnitzer JJ, Wilson JM. Insulin-like growth factor-I gene expression in three models of accelerated lung growth. J Pediatr Surg 1998;33:1057–1060.[CrossRef][Medline]
  25. Price WA, Moats-Staats BM, Sekhon HS, Chrzanowska BL, Thurlbeck WM, Stiles AD. Expression of the insulin-like growth factor system in postpneumonectomy lung growth. Exp Lung Res 1998;24:203–217.[Medline]
  26. Sugahara K, Matsumoto M, Baba T, Nakamura T, Kawamoto T. Elevation of serum human hepatocyte growth factor (HGF) level in patients with pneumonectomy during a perioperative period. Intensive Care Med 1998;24:434–437.[CrossRef][Medline]
  27. Wandel G, Berger LC, Burri PH. Morphometric analysis of adult rat lung after bilobectomy. Am Rev Respir Dis 1983;128:968–972.[Medline]
  28. Foster DJ, Moe OW, Hsia CC. Upregulation of erythropoietin receptor during postnatal and postpneumonectomy lung growth. Am J Physiol Lung Cell Mol Physiol. 2004;287:L1107–L1115.[Abstract/Free Full Text]
  29. Mineo TC, Ambrogi V, Baldi A, Rabitti C, Bollero P, Vincenzi B, Tonini G. Prognostic impact of VEGF, CD31, CD34, and CD105 expression and tumour vessel invasion after radical surgery for IB-IIA non-small cell lung cancer. J Clin Pathol 2004;57:591–597.[Abstract/Free Full Text]
  30. Le Cras TD, Fernandez LG, Pastura PA, Laubach VE. Vascular growth and remodeling in compensatory lung growth following right lobectomy. J Appl Physiol 2005;98:1140–1148.[Abstract/Free Full Text]
  31. Folkman J, Klagsbrun M. Angiogenic factors. Science 1987;235:442–447.[Abstract/Free Full Text]
  32. Folkman J, D'Amore PA. Blood vessel formation: what is its molecular basis? Cell 1996;87:1153–1155.[CrossRef][Medline]
  33. Thomas KA. Vascular endothelial growth factor, a potent and selective angiogenic agent. J Biol Chem 1996;271:603–606.[Free Full Text]
  34. Wang M, Bronte V, Chen PW, Gritz L, Panicali D, Rosenberg SA, Restifo NP. Active immunotherapy of cancer with a nonreplicating recombinant fowlpox virus encoding a model tumor-associated antigen. J Immunol 1995;154:4685–4692.[Abstract]
  35. Kong HL, Hecht D, Song W, Kovesdi I, Hackett NR, Yayon A, Crystal RG. Regional suppression of tumor growth by in vivo transfer of a cDNA encoding a secreted form of the extracellular domain of the flt-1 vascular endothelial growth factor receptor. Hum Gene Ther 1998;9:823–833.[Medline]
  36. Hersh J, Crystal RG, Bewig B. Modulation of gene expression after replication-deficient, recombinant adenovirus-mediated gene transfer by the product of a second adenovirus vector. Gene Ther 1995;2:124–131.[Medline]
  37. Rosenfeld MA, Siegfried W, Yoshimura K, Yoneyama K, Fukayama M, Stier LE, Paakko PK, Gilardi P, Stratford-Perricaudet LD, Perricaudet M, et al. Adenovirus-mediated transfer of a recombinant alpha 1-antitrypsin gene to the lung epithelium in vivo. Science 1991;252:431–434.[Abstract/Free Full Text]
  38. Rosenfeld MA, Yoshimura K, Trapnell BC, Yoneyama K, Rosenthal ER, Dalemans W, Fukayama M, Bargon J, Stier LE, Stratford-Perricaudet L, et al. In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium. Cell 1992;68:143–155.[CrossRef][Medline]
  39. Crystal RG, McElvaney NG, Rosenfeld MA, Chu CS, Mastrangeli A, Hay JG, Brody SL, Jaffe HA, Eissa NT, Danel C. Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat Genet 1994;8:42–51.[CrossRef][Medline]
  40. Mastrangeli A, Danel C, Rosenfeld MA, Stratford-Perricaudet L, Perricaudet M, Pavirani A, Lecocq JP, Crystal RG. Diversity of airway epithelial cell targets for in vivo recombinant adenovirus-mediated gene transfer. J Clin Invest 1993;91:225–234.
  41. Haasler F. Über compensatorische hypertrophie der Lunge. Virochows Arch Pathol Anat Pathol Anat Physiol. 1892;128:527–536.
  42. Benedict JH, Rannels DE. Postpneumonectomy lung growth following thyroparathyroidectomy. Exp Lung Res 1994;20:13–25.[Medline]
  43. Brody JS. Time course of and stimuli to compensatory growth of the lung after pneumonectomy. J Clin Invest 1975;56:897–904.
  44. Doerschuk CM, Sekhon HS. Pulmonary blood volume and edema in postpneumonectomy lung growth in rats. J Appl Physiol 1990;69:1178–1182.[Abstract/Free Full Text]
  45. Dubaybo BA, Bayasi G, Rubeiz GJ. Changes in tumor necrosis factor in postpneumonectomy lung growth. J Thorac Cardiovasc Surg 1995;110:396–404.[Abstract/Free Full Text]
  46. Koh DW, Roby JD, Starcher B, Senior RM, Pierce RA. Postpneumonectomy lung growth: a model of reinitiation of tropoelastin and type I collagen production in a normal pattern in adult rat lung. Am J Respir Cell Mol Biol 1996;15:611–623.[Abstract]
  47. Nattie EE, Wiley CW, Bartlett D. Adaptive growth of the lung following pneumonectomy in rats. J Appl Physiol 1974;37:491–495.[Free Full Text]
  48. Thet LA, Law DJ. Changes in cell number and lung morphology during early postpneumonectomy lung growth. J Appl Physiol 1984;56:975–978.[Abstract/Free Full Text]
  49. American Thoracic Society, ad hoc Statement Committee. Mechanisms and limits of induced postnatal lung growth. 2004. Am J Respir Crit Care Med.170:319–343.[Free Full Text]
  50. Folkman J, Cole P, Zimmerman S. Tumor behavior in isolated perfused organs: in vitro growth and metastases of biopsy material in rabbit thyroid and canine intestinal segment. Ann Surg 1966;164:491–502.[Medline]
  51. Folkman J. Incipient angiogenesis. J Natl Cancer Inst 2000;92:94–95.[Free Full Text]
  52. Liu W, Ahmad SA, Reinmuth N, Shaheen RM, Jung YD, Fan F, Ellis LM. Endothelial cell survival and apoptosis in the tumor vasculature. Apoptosis 2000;5:323–328.[CrossRef][Medline]
  53. Kendall RL, Thomas KA. Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor. Proc Natl Acad Sci USA 1993;90:10705–10709.[Abstract/Free Full Text]
  54. Kendall RL, Wang G, Thomas KA. Identification of a natural soluble form of the vascular endothelial growth factor receptor, FLT-1, and its heterodimerization with KDR. Biochem Biophys Res Commun 1996;226:324–328.[CrossRef][Medline]
  55. Kodama M, Kodama T, Nishi Y, Totani R. Does surgical stress cause tumor metastasis? Anticancer Res 1992;12:1603–1616.[Medline]
  56. Davidson RS, Nwogu CE, Brentjens MJ, Anderson TM. The surgical management of pulmonary metastasis: current concepts. Surg Oncol 2001;10:35–42.[CrossRef][Medline]




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