Published OnlineFirst August 28, 2016; DOI: 10.1158/0008-5472.CAN-16-1131
Cancer
Research
Integrated Systems and Technologies
Bronchial Artery Angiogenesis Drives Lung
Tumor Growth
Lindsey Eldridge, Aigul Moldobaeva, Qiong Zhong, John Jenkins, Michael Snyder,
Robert H. Brown, Wayne Mitzner, and Elizabeth M. Wagner
Abstract
Angiogenesis is vital for tumor growth but in well-vascularized organs such as the lung its importance is unclear. This
situation is complicated by the fact that the lung has two
separate circulations, the pulmonary and the systemic bronchial circulation. There are few relevant animal models of non–
small cell lung cancer, which can be used to study the lung's
complex circulations, and mice, lacking a systemic bronchial
circulation cannot be used. We report here a novel orthotopic
model of non–small cell lung cancer in rats, where we have
studied the separate contributions of each of the two circulations for lung tumor growth. Results show that bronchial artery
perfusion, quantified by fluorescent microspheres (206%
increase in large tumors) or high-resolution computed tomography scans (276% increase in large tumors), parallels the
growth in tumor volume, whereas pulmonary artery perfusion
remained unchanged. Ablation of the bronchial artery after the
initiation of tumor growth resulted in a decrease in tumor
volume over a subsequent course of 4 weeks. These results
demonstrate that although the existing pulmonary circulation
can supply the metabolic needs for tumor initiation, further
growth of the tumor requires angiogenesis from the highly
proliferative bronchial circulation. This model may be useful to
investigate new therapeutic approaches that target specifically
the bronchial circulation. Cancer Res; 76(20); 1–8. 2016 AACR.
Introduction
capacity for angiogenesis, the proliferating bronchial vasculature provides the primary source for new vessels and subsequently, tumor growth. The vascular supply of solid lung
tumors has not been well studied, because there are few good
animal models of lung cancer.
This lack of a good lung cancer model has limited mechanistic studies of the vascular sources, in part due to the
difficulty of separating and measuring pulmonary versus bronchial perfusion. Also, because of the difficulty of taking multiple serial measurements tracking tumor size and perfusion,
studies in humans with lung cancer have only measured
perfusion associated with a single tumor volume, providing
no information about the temporal changes to each vascular
bed with increasing tumor size. Common xenograft models in
rodents using the flank lack the circulatory complexity of the
lung, and mouse lung models are inadequate due to the fact
that mice lack a subcarinal bronchial vasculature (10). A
further complicating factor is that in the process of tumor
vascular growth, new anastomoses can form between the
bronchial and pulmonary circulations. So even if the bronchial
circulation is primary, such anastomoses could possibly allow
the pulmonary circulation to supply a secondary nutrient flow
to the tumor. Indeed, there is radiographic evidence suggesting
that pulmonary anastomoses can supply metastatic lung
tumors (11). Each of these methodologic challenges has been
an obstacle to mechanistic studies defining the dual circulations involved in lung tumor perfusion.
In this article, we describe a novel rat model of NSCLC and
quantify bronchial and pulmonary tumor perfusion independently as they change with tumor growth. We show that
impairment of the bronchial circulation cannot only stop tumor
growth, but also may lead to diminishing tumor size. This model
opens new therapeutic possibilities for studying treatment of
primary lung cancer.
For tumor growth, new blood vessels are necessary if a
tumor is to grow to any substantial size beyond that which
can be supplied by simple diffusion from the existing circulation (1). Although this vascular need in tumors has been
clearly demonstrated in systemic tissues (2), how it manifests
itself in the lung is not entirely clear. The lung is unique
because it has two distinct sources of blood; the deoxygenated
pulmonary circulation and the systemic bronchial circulation
that derives from the aorta. One might imagine that with the
pulmonary circulation providing a conduit for the entire
cardiac output, there would be more than sufficient blood
flow for tumor growth. In fact, in non–small cell lung cancer,
there have been some reports of nonangiogenic tumors sustaining themselves by vascular co-option (3, 4). However,
there is good evidence that the pulmonary circulation has very
limited angiogenic capacity, whereas the bronchial circulation
has been shown to have prolific and substantial angiogenic
properties (5, 6). Indeed, there are several studies that clearly
show a preference for the bronchial circulation by lung tumors
(7–9). It may be that the pulmonary vasculature can function
as a maintenance vasculature for small tumors but, with little
Departments of Medicine and Environmental Health Sciences, Johns
Hopkins University, Baltimore, Maryland.
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Author: Elizabeth M. Wagner, Johns Hopkins University, 5501
Hopkins Bayview Circle, Baltimore, MD 21224. Phone: 410-550-2506; Fax: 410550-2612; E-mail: [email protected]
doi: 10.1158/0008-5472.CAN-16-1131
2016 American Association for Cancer Research.
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Eldridge et al.
Materials and Methods
In vitro determination of angiogenic potential of endothelial
cells
Endothelial cell isolation. Primary endothelial cells were isolated
from the pulmonary artery (PAEC) and pulmonary microvasculature (MVEC) using methods described previously (12). The
bronchial artery was visualized on the dorsal surface of the trachea
and main stem bronchus and dissected. Bronchial artery endothelial cells (BAEC) were isolated following the same protocol.
Cells were maintained in 2% FBS cultures under basal conditions
and were studied between passage 3 and 5.
FACS analysis of endothelial cells. To determine proliferation after
a 24-hour incubation period with adenocarcinoma supernatant, endothelial cells were stained with thrombomodulin
(anti-CD141; Bioss Antibodies) and live cells were selected
using VIVID (Invitrogen). For the cell proliferation marker,
cells were incubated with fixation/permeabilization buffer
(eBiosciences) and intracellularly stained with fluorescencelabeled anti-Ki67, an antigen found in all active phases of the
cell cycle (BD Pharmingen). Sample profiles were acquired on a
BD FACSaria (BD) and data were analyzed with FlowJo Software (TreeStar).
Chemotaxis. BAECs, PAECs, and MVECs were cultured in the top
chamber of transwell plates (6.5-mm diameter inserts, 5.0-mm
pore size polycarbonate transwell membrane; Corning Incorporated) in 2% FBS, whereas adenocarcinoma supernatant was
added to the bottom chamber. Cells in the bottom chamber
were counted using a hemocytometer after 24 hours of
incubation.
Endothelial tube formation. BAECs, PAECs, and MVECs were
grown in a 3D culture (Matrigel, growth factor reduced basement
membrane matrix; Corning Incorporated) with adenocarcinoma
supernatant for 24 hours. Each endothelial cell type, from nine
different animals, was cultured in triplicate wells. Images from
three fields of view per well were taken at 100 original magnification (Olympus IX51 microscope and High Performance
SensiCam), and total tube lengths of connecting cells from the
three images were measured by use of Image Pro Plus 5.1 software (Media Cybernetics).
In vivo lung adenocarcinoma model
Adenocarcinoma cell delivery. The experimental protocol was
approved by the Johns Hopkins Animal Care and Use Committee
(Protocol #RA12M283). Human lung adenocarcinoma cells
(Calu-3, ATCC; tested and authenticated by vendor's cytogenetic
analysis) were grown in culture, and 5 106 cells in 50 mL of sterile
saline were injected transthoracically (without thoracotomy) into
the left lung of anesthetized, ventilated RNU nude rats (Charles
River Laboratories).
Tumor volume by high-resolution computed tomography scan. Rats
were anesthetized with a ketamine/xylazine (75/25 mg/kg) solution. High-resolution computed tomography (HRCT) scans were
performed in the Department of Radiology, CT, at the Johns
Hopkins Outpatient Center, on a Seimens Definition Flash 256
slice dual source scanner. Data analysis and scanner specifications
are described in Supplementary Fig. S1.
OF2 Cancer Res; 76(20) October 15, 2016
Tumor perfusion by fluorescent microsphere injection. Differential
tumor perfusion from the bronchial versus the pulmonary circulation was determined using two different fluorescent microsphere colors. Surgical procedures, tissue digestion, and fluorescence quantification were described previously (13). In addition
to the previously described protocol for the bronchial circulation,
a femoral vein catheter was infused for quantification of pulmonary perfusion (106, 10 mL yellow polystyrene fluorescent microspheres; Invitrogen). After exsanguination, lung tumors were
visualized and carefully dissected from lung tissue for digestion
and fluorescence analysis of crimson (bronchial) and yellow
(pulmonary) microspheres.
Tumor perfusion by contrast-enhanced HRCT. Rats were anesthetized and a catheter was placed in a femoral vein for contrast
(Visipaque 320; GE Healthcare) infusion. Image analysis and
scanner settings can be found in Supplementary Fig. S2.
Bronchial artery ablation. After an initial measurement of tumor
volume by HRCT (4 weeks), rats were anesthetized, ventilated,
and a thoracotomy was performed. The left lung was repositioned
with forceps and the airway was exposed to visualize bronchial
arteries on the dorsal surface of the left main stem bronchus.
Bronchial arteries were cauterized using a Gemini Cautery System
(Braintree Scientific). In sham animals, the same surgical procedure was followed, but an intercostal artery was cauterized instead
of the bronchial artery. The lung was expanded and the thorax
closed.
Statistical analysis
All data are presented as the mean SE. A one-way ANOVA
with Bonferroni's multiple comparison test was used to evaluate
proliferation, chemotaxis, and tube formation. Log-transformed
regressions were performed to measure the correlations between
tumor weight and number of microspheres as well as tumor
volume and voxel intensity. t tests were used to compare perfusion
and volume between small and large tumor groups. A P value 0.05 was accepted as significant.
Results
In vitro determination of angiogenic potential of endothelial
cells
BAECs, PAECs, and MVECs were stimulated with adenocarcinoma supernatant to determine the angiogenic potential of
each endothelial cell type. When quantifying Ki67þ staining as
a measurement of proliferation, basal rates of proliferation
varied among cell type and between experiments (2–15%
Ki67þ) but showed no overall group differences. After normalization to average baseline proliferation for each cell type,
exposure to adenocarcinoma supernatant resulted in significantly increased levels of proliferation in BAECs (P < 0.0001)
when compared with both PAECs and MVECs (Fig. 1A). Under
basal conditions, there was no chemotaxis among any cell type.
Yet BAECs showed significantly greater chemotaxis in response
to adenocarcinoma supernatant than PAECs (P ¼ 0.04; Fig. 1B).
In 3D Matrigel cultures, there was little tube formation in any
cell type under basal conditions. However, upon supernatant
stimulation, the sum of tube lengths (mm) was significantly
greater in BAEC cultures than PAEC and MVEC (P < 0.001; Fig.
1C). To further confirm the angiogenic potential of each
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Lung Tumor Angiogenesis
***
4
2
*
BAEC
PAEC
MVEC
400
300
200
100
BAEC
***
15,000
0
0
Tube formation
C
Chemotaxis
500
Number of cells
6
% Ki67+ Staining
B
Proliferation
***
Total tube lengths (μm)
A
PAEC
MVEC
**
10,000
5,000
0
BAEC
PAEC
MVEC
Figure 1.
Adenocarcinoma culture supernatant stimulation of BAECs, PAECs, and MVEC. A, BAECs showed significantly increased levels of proliferation by Ki67þ staining
compared with PAECs and MVECs ( , P < 0.0001). B, BAECs showed increased chemotaxis compared with PAECs ( , P ¼ 0.04). C, BAECs showed
significantly more tube formation (S of tube lengths) than either PAECs ( , P < 0.0001) or MVECs ( , P < 0.001) in response to adenocarcinoma culture supernatant.
endothelial cell type, VEGF protein, one growth factor, known
to be secreted by adenocarcinoma cells (14, 15), was used for
stimulation in each experimental setting. When endothelial
cells were stimulated with VEGF (10 ng/mL), a significant
increase in proliferation (83% increase compared with both
PAECs and MVECs; n ¼ 9, P ¼ 0.0005) and tube formation
(94% and 93% increases compared to PAECs and MVECs,
respectively; n ¼ 6, P < 0.0001) were observed. The results of
these in vitro assessments of angiogenic potential demonstrated
a markedly enhanced responsiveness in isolated BAECs relative
to PAECs and MVECs.
In vivo lung adenocarcinoma model
The model we use involves direct injections of adenocarcinoma
cells through the chest wall into the left lung. We used different
techniques as described below to measure the growth of tumor
mass and perfusion as a function of time after the injection. These
measurements were also made after ablation of the bronchial
artery.
Tumor mass by HRCT. To confirm the presence of tumor formation
after adenocarcinoma cell injection, HRCT scans were performed
weekly after the delivery of adenocarcinoma cells directly into the
left lung. Representative thoracic HRCT images from a single rat
depict four time points after adenocarcinoma cell injection
(Fig. 2A). In this specific rat, tumor detection was not possible
until 3 weeks after adenocarcinoma cell injection. However, in
other rats, tumors were detected as early as 2 weeks. The HRCT
scans at 4 and 8 weeks demonstrate the progressive increase in
tumor size over time. Tumor volume was calculated from the
summation of the serial regions of interest (white outline) on the
image slices containing the tumor (see Supplementary Methods).
The growth patterns observed in Fig. 2B depict a progressive
increase in tumor volume (cm3) over the course of 8 weeks,
although the rate of growth varied among animals (A–F; Fig.
2B). To confirm the validity of HRCT to quantify tumor mass, in a
separate series of seven rats, tumor volume measured by HRCT
was shown to significantly correlate with tumor weight (r2 ¼ 0.98,
P < 0.0001; Fig. 2C).
Tumor perfusion by microsphere injection. Fluorescent microsphere
injection was used to quantify tumor perfusion from the pulmonary or bronchial circulations. By using two different colored
microspheres and dual injection sights (carotid artery to aorta:
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representing the bronchial circulation, and femoral vein to pulmonary artery: representing the pulmonary circulation), the contribution of each circulation to total tumor perfusion was quantified. The total number of microspheres counted in the tumor
(bronchial þ pulmonary perfusion) significantly increased with
tumor weight (r2 ¼ 0.541; P ¼ 0.0018; n ¼ 15; Fig. 3A). When
tumor weights from all rats were dichotomized to less than or
greater than 0.5 g, the larger tumors had a significantly higher
percentage of microspheres from the bronchial circulation than
the smaller tumors (P ¼ 0.003; Fig. 3B). The pulmonary circulation did not change its contribution to tumor perfusion between
small and large tumors.
Tumor perfusion by contrast-enhanced HRCT scanning. Contrastenhanced HRCT scans were used to independently measure in vivo
tumor perfusion [intensity of contrast medium in Hounsfield
units (HU) as a fraction of maximum HU; see Supplementary
Methods]. Contrast HRCT scanning confirmed that bronchial
perfusion significantly increased with tumor volume (r2 ¼ 0.74;
P ¼ 0.0003; n ¼ 11; Fig. 4A). When tumors were dichotomized
to less than or greater than 0.3 cm3 in volume, there was
significantly greater bronchial perfusion to the larger tumors
(P < 0.0001; Fig. 4B). Consistent with previous results, there
was no difference in pulmonary perfusion as measured by
contrast-enhanced HRCT (Fig. 4C), and when tumors were
partitioned by volume (<0.3 vs. >0.3 cm3) there was no significant difference in pulmonary perfusion between small and
large tumors (Fig. 4D).
Tumor volume after bronchial artery ablation. To confirm the
importance of bronchial vascular angiogenesis in lung tumor
growth, the bronchial artery was cauterized 4 weeks after adenocarcinoma cell injection. HRCT was used to determine initial
tumor volume in rats 4 weeks after injection of adenocarcinoma
cells, and at final tumor volume at 8 weeks. Rats were divided into
two groups; one group with an intact bronchial artery (intact BA;
n ¼ 8) and a second group where the bronchial artery was
cauterized the day after the initial tumor volume scan (ablated
BA; n ¼ 7). Two animals (represented by triangles) were sham
controls that followed the same surgical procedures as the "ablated BA" group, except an intercostal artery was cauterized instead of
the bronchial artery. Figure 5A shows the individual tumor
volumes in rats with an intact bronchial artery and in the group
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Eldridge et al.
3 wk
Time: 2 wk
A
L
R
B
8 wk
4 wk
R
L
L
R
C
2.0
L
R
1.5
B
1.5
C
Weight (g)
Tumor volume (cm3)
A
D
1.0
E
F
1.0
0.5
0.5
0.0
0.0
0.0
1
2
3
4
5
6
7
8
0.5
1.0
1.5
Tumor volume (cm3)
Week
Figure 2.
Orthotopic lung adenocarcinoma model in rat. A, thoracic HRCT images from a single rat at four time points after injection of adenocarcinoma cells (see
Materials and Methods). At week 3, a single solid adenocarcinoma tumor can be seen in the left lung. (note the adenocarcinoma outlined in white as the region of
interest.) The HRCT scans at weeks 4 and 8 demonstrate the progressive increase in tumor size over time. Tumor volume was calculated from the summation
of the serial regions of interest (white outline) on the image slices containing the tumor (see Supplementary Methods). B, demonstrates the progressive increase
in tumor volume (cm3) in each rat as determined by HRCT scans over the course of 8 weeks. Each line represents a single tumor in different individual rats
(n ¼ 6; rat B depicted in CT scan). C, in a separate series of rats (n ¼ 7), tumor volume as measured by HRCT was significantly correlated with tumor weight by
pathology (r2 ¼ 0.98, P < 0.0001). These results demonstrate the accuracy of the HRCT scanning method to measure tumor size.
with an ablated BA. There was significantly less change in tumor
volume between 4 and 8 weeks in animals after bronchial artery
ablation (P ¼ 0.0009; Fig. 5B) and in absolute tumor volume at 8
weeks. The tumor volume was significantly decreased in rats after
BA ablation (P ¼ 0.0076; Fig. 5C). Contrast-enhanced HRCT scans
were performed at the time of the 8-week tumor volume measurement to confirm the sustained reduction in bronchial tumor
perfusion resulting from the bronchial artery cauterization. In
animals with BA ablation, there was a significant decrease in both
bronchial tumor perfusion and the slope of the relationship
Discussion
Folkman first described the vital role angiogenesis plays in
tumor growth and suggested that inhibiting it would lead to
tumor necrosis and cancer cell death (2). However, the manifestation of this conjecture has been inconsistent since that time. In
particular, in the lung, it is possible for a growing tumor to utilize
B
30,000
**
40
% Bronchial perfusion
Total number of microspheres
A
between tumor volume and bronchial perfusion compared with
intact BA.
20,000
10,000
0
30
20
10
0
0
1
2
Tumor weight (g)
OF4 Cancer Res; 76(20) October 15, 2016
3
<0.5 g
>0.5 g
Tumor weight (g)
Figure 3.
Tumor perfusion measured by
microsphere injection. A, the total
number of microspheres counted in the
tumor (bronchial þ pulmonary
perfusion) was significantly correlated
with tumor weight (r2 ¼ 0.541;
P ¼ 0.0018). B, when tumors were
partitioned into two groups by weight
(<0.5 g vs. >0.5 g), the larger tumors
had a significantly higher percentage of
microspheres from the bronchial
circulation than the small tumors
(P ¼ 0.003).
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Lung Tumor Angiogenesis
Bronchial perfusion
B
Intensity (HU/max HU)
0.06
0.04
0.02
0.00
0.0
0.06
***
0.04
0.02
0.00
0.5
1.0
<0.3 cm 3
1.5
Tumor volume (cm3)
>0.3 cm 3
Tumor volume (cm3)
Pulmonary perfusion
D
0.10
Intensity (HU/max HU)
C
Intensity (HU/max HU)
Figure 4.
Tumor perfusion determined by
contrast enhanced HRCT scanning. A,
tumor perfusion (intensity of contrast
medium HU as a fraction of maximum
HU, also see Supplementary Methods)
from the bronchial circulation was
significantly correlated with tumor
volume (r2 ¼ 0.74; P ¼ 0.003; n ¼ 11).
The larger the tumor, the greater was
the bronchial perfusion as measured
on contrast enhanced HRCT. B, when
tumors were partitioned by volume
(<0.3 cm3 vs. >0.3 cm3), there was a
significantly greater bronchial
perfusion to the larger tumors
( , P < 0.0001). C, in contrast, there
was no difference in pulmonary
perfusion as measured by contrastenhanced HRCT by tumor volume. D,
when tumors were partitioned by
volume (<0.3 cm3 vs. >0.3 cm3), there
was no significant difference in
pulmonary perfusion between small
and large tumors.
Intensity (HU/max HU)
A
0.08
0.06
0.04
0.02
0.00
0.0
0.06
0.04
0.02
0.00
0.5
1.0
1.5
Tumor volume (cm3)
the existing vasculature by vessel co-option, suggesting neovascularization is not always necessary for tumor establishment and
growth (3, 16). However, given the diminished capacity for the
pulmonary circulation to undergo angiogenesis (17), a solid
tumor might outgrow what can be supplied by the pulmonary
circulation, whereupon it would then require its own additional
A
<0.3 cm3
>0.3 cm3
Tumor volume (cm3)
blood supply. The dynamics of this process in conjunction with
tumor growth in the lung have not been studied. Several groups
have utilized contrast-enhanced HRCT studies in patients to
quantify functional tumor perfusion (8, 18, 19). Yuan and colleagues introduced refined CT perfusion protocols in a small study
of patients with diverse primary lung carcinomas and showed that
Intact BA
Ablated BA
1.5
1.0
0.5
0.0
4 Weeks
B
8 Weeks
Change in tumor volume 4–8 weeks
C
8 Weeks
Tumor volume at 8 weeks
**
Tumor volume (cm 3)
1.5
1.0
0.5
0.0
1.0
0.5
0.0
–0.5
Intact BA
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4 Weeks
**
1.5
Δ Tumor volume (cm3)
Figure 5.
Tumor volume assessed by HRCT
scans 4 weeks after bronchial artery
cauterization. A, tumor volume was
determined in rats 4 weeks after
injection of adenocarcinoma and
again at 8 weeks in rats with an intact
bronchial artery (intact BA; n ¼ 8) and
in an additional group where the
bronchial artery was cauterized the
day after the initial scan (ablated BA;
n ¼ 7). B, there was a significant
attenuation in tumor volume growth
between 4 and 8 weeks in animals that
had bronchial artery ablation
compared with animals with intact
bronchial arteries ( , P < 0.001).
C, at 8 weeks, tumor volume was
significantly decreased in rats after BA
ablation ( , P ¼ 0.0076).
Tumor volume (cm 3)
2.0
Ablated BA
Intact BA
Ablated BA
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tumor angiogenesis was somewhat size-dependent overall (7),
but the systemic circulation played the dominant role. However,
longitudinal tracking information of tumor volumes with vascularization in individual subjects is not available in patients or
animal models. This study was undertaken to define the parallel
processes of solid tumor growth in the lung and the corresponding
angiogenesis, while simultaneously identifying the specific supporting vasculature.
Essential to this study was a physiologically relevant animal
model that would allow for longitudinal measurement of tumor
volumes and perfusion sources. We developed a model of NSCLC
in nude rats, resulting in the development of a single solid tumor.
Substantial tumor growth took place over the course of 8 weeks, as
confirmed by tumor weight, tumor volume by HRCT, and lung
pathology. Histologic sections showed clear separation between
proliferating tumor and normal lung parenchyma, with additional necropsy confirming the absence of any systemic metastases.
Once the model was established, the source of tumor perfusion in
this rat model could be quantified. It is worth noting that such a
study of the lung tumor vascular source cannot be done in mouse
models. Mice lack a subcarinal bronchial artery (10, 20), so
cannot be used to model the source of tumor perfusion in
humans. Consequently, the results of several mouse models
including Lewis lung carcinomas (21), those with the overexpression of oncogenes (22–24), or of xenografts in the flank (25, 26)
may have limited applicability to the central question of perfusion
pathology.
In our study, we provide two functional measurements with
complementary information concerning the source and proliferation of the vasculature supplying the growing tumor. Different
colors of fluorescent microspheres infused separately into the
pulmonary circulation and the systemic bronchial circulation
confirmed the ever-increasing systemic perfusion of the growing
tumor. In small tumors, perfusion from the bronchial circulation
was less than 10% of total tumor perfusion. However, in larger
tumors, bronchial perfusion increased significantly to an average
30.6% of total tumor perfusion (average 206% increase). Because
these were terminal experiments with only one tumor weight
measured at completion of the experiment to pair with microsphere perfusion, the use of HRCT to monitor longitudinal tumor
growth provided important additional information. With perfusion HRCT, a similar overall change in perfusion was measured. In
small tumors, systemic bronchial perfusion constituted approximately 21% of total perfusion. Beyond the 0.3 cm3 median size of
tumors studied, bronchial perfusion increased an average of
276%. Differences in magnitude of the initial perfusion with the
two methods likely can be attributed to different tumor sizing
methods (weight vs. calculated volume) and different median
sizes for the analysis. Nevertheless, the changes in bronchial
perfusion with the two different functional measurements were
quite similar in the magnitude of increased perfusion (206% vs.
276%). Importantly, both perfusion measurements demonstrated that the pulmonary circulation remained relatively constant
and did not increase with tumor size. These functional results
demonstrate that the bronchial circulation undergoes angiogenesis to support the needs of the growing lung tumor over the
course of 8 weeks, whereas the pulmonary vasculature remains
unchanged.
To further confirm the critical role of bronchial angiogenesis in
tumor growth, we surgically intervened and studied subsequent
tumor growth after the elimination of the bronchial circulation.
OF6 Cancer Res; 76(20) October 15, 2016
After 4 weeks of tumor growth, the left bronchial artery was
cauterized. After this ablation, there was no further change in
tumor volume for at least 4 weeks after the surgical intervention.
Final tumor volumes at 8 weeks were significantly smaller than the
8-week tumor volumes in animals with an intact bronchial artery
(Fig. 5). Perfusion measurements made at the corresponding 8week tumor volume determination confirmed the continued
absence of any new systemic blood flow, as determined by the
significant decrease in the both the baseline and slope of the
relationship between bronchial perfusion and tumor size. Thus,
angiogenesis of the bronchial circulation is critical for tumor
growth. Without new blood vessels supplied by the systemic
bronchial vasculature, lung tumors could not grow beyond the
minimal size, which could be supported by the pulmonary
circulation alone.
The results of bronchial artery ablation should be put in context
with studies in humans attempting to modify this vascular source.
As early as 1969, bronchial artery infusion therapy was used as a
route for more direct delivery of chemotherapy to lung tumors in
patients with lung cancer (27), and such approaches to treat
NSCLC and hepatocellular carcinoma lung metastases are still
being used (28, 29). It is also common for patients with NSCLC to
experience life-threatening hemoptysis, and for decades it has
been routine practice to embolize small branches of the bronchial
artery as an effective treatment (30, 31). In cases where bronchial
artery embolization has been performed, there have been no
reports on how this procedure specifically alters tumor growth.
Because only small branches of the bronchial artery are typically
embolized and patients generally are also undergoing chemotherapy, results of embolization per se on lung tumor growth have
not been established. It appears that a more complete bronchial
artery ablation has yet to be used as a therapeutic option in
advanced disease states.
Although the focus of this study was not to identify relevant
growth factors required for the process of bronchial vascular
angiogenesis, our initial observations regarding bronchial endothelium provide some insights. Bronchial endothelial cells
showed robust proliferative characteristics, chemotaxis, and tube
formation in response to both adenocarcinoma supernatant and
VEGF, compared with pulmonary macro- or microvascular endothelium. As these in vitro measurements are frequently used as
exclusive indicators of angiogenesis, the interpretation of many
previous studies where examination of relevant endothelial cell
types was not considered becomes very difficult. Given the focus
of many cancer therapies on tumor angiogenesis (32–34), recognizing and exploiting differences in endothelium seems essential.
Although this study was designed to examine the vascular
contributions to a solid NSCLC tumor, it is unknown whether
the results might be extrapolated to other lung cancer cell types.
Small cell carcinoma or bronchial carcinoma may have different
perfusion patterns dependent on tumor size and location. It is
reasonable to suggest using this model with the injection of a
small cell carcinoma, or xenograft obtained directly from patient
samples, to further examine tumor perfusion patterns. Clinically,
contrast-enhanced CT scanning could be performed in patients
with NSCLC to determine the perfusion contribution from the
bronchial circulation, therefore dictating subsequent treatment
protocols. Given how effective bronchial artery ablation was in
limiting tumor growth in our model, it might be expected to
similarly compromise lung tumor growth in patients and other
models of lung cancer. Of course, from a potential therapeutic
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perspective, limiting the total systemic arterial perfusion to an
organ may hurt the organ as much as the tumor. So only tumors in
the liver with its two circulations would be very relevant to our
present model in the lung. Indeed, it is known that the systemic
hepatic circulation provides the primary blood flow to liver
tumors (28), much like the bronchial circulation being essential
for large tumors in the lung parenchyma as shown here.
Conclusion
Tumors have been shown to utilize several methods for vascularization including angiogenesis and vessel co-option. Given
the extensive pulmonary capillary network, lung tumors may
initially not require extensive angiogenesis. However, if the tumor
is to grow more massive, the results of this study demonstrate the
essential role for the proliferative capacity of bronchial endothelium compared with the pulmonary vasculature. Tumor growth
required bronchial angiogenesis, so targeting the bronchial endothelium may provide opportunities to develop new therapeutic
approaches.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: L. Eldridge, R.H. Brown, W. Mitzner, E.M. Wagner
Development of methodology: L. Eldridge, R.H. Brown, W. Mitzner,
E.M. Wagner
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): L. Eldridge, J. Jenkins, M. Snyder, R.H. Brown,
W. Mitzner, E.M. Wagner
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): L. Eldridge, M. Snyder, R.H. Brown, W. Mitzner,
E.M. Wagner
Writing, review, and/or revision of the manuscript: L. Eldridge, J. Jenkins,
M. Snyder, R.H. Brown, W. Mitzner, E.M. Wagner
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): L. Eldridge, A. Moldobaeva, Q. Zhong, J. Jenkins,
W. Mitzner
Study supervision: E.M. Wagner
Acknowledgments
The authors acknowledge NHLBI for supporting this work (HL10342 and
HL113392).
Grant Support
This work was supported by NHLBI grants HL10342 and HL113392.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received April 25, 2016; revised July 25, 2016; accepted August 11, 2016;
published OnlineFirst August 28, 2016.
References
1. Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston.
Clinical applications of research on angiogenesis. N Engl J Med
1995;333:1757–63.
2. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med
1971;285:1182–6.
3. Pezzella F, Pastorino U, Tagliabue E, Andreola S, Sozzi G, Gasparini G, et al.
Non-small-cell lung carcinoma tumor growth without morphological
evidence of neo-angiogenesis. Am J Pathol 1997;151:1417–23.
4. Offersen BV, Pfeiffer P, Hamilton-Dutoit S, Overgaard J. Patterns of
angiogenesis in nonsmall-cell lung carcinoma. Cancer 2001;91:1500–9.
5. Charan NB, Carvalho P. Angiogenesis in bronchial circulatory system after
unilateral pulmonary artery obstruction. J Appl Physiol 1997;82:284–91.
6. Michel RP, Hakim TS, Petsikas D. Segmental vascular resistance in postobstructive pulmonary vasculopathy. J Appl Physiol 1990;69:1022–32.
7. Yuan X, Zhang J, Ao G, Quan C, Tian Y, Li H. Lung cancer perfusion: can we
measure pulmonary and bronchial circulation simultaneously? Eur Radiol
2012;22:1665–71.
8. Nguyen-Kim TD, Frauenfelder T, Strobel K, Veit-Haibach P, Huellner MW.
Assessment of bronchial and pulmonary blood supply in non-small cell
lung cancer subtypes using computed tomography perfusion. Invest Radiol
2015;50:179–86.
9. Nguyen KS, Neal JW, Wakelee H. Review of the current targeted therapies
for non-small-cell lung cancer. World J Clin Oncol 2014;5:576–87.
10. Verloop MC. On the arteriae bronchiales and their anastomosing with the
arteria pulmonalis in some rodents; a micro-anatomical study. Acta Anat
1949;7:1–32.
11. Milne EN, Zerhouni EA. Blood supply of pulmonary metastases. J Thorac
Imaging 1987;2:15–23.
12. Moldobaeva A, Baek A, Wagner EM. MIP-2 causes differential activation of
RhoA in mouse aortic versus pulmonary artery endothelial cells. Microvasc
Res 2008;75:53–8.
13. Jenkins J, Wagner E. Angiogenesis in the ischemic rat lung. J Vis Exp; 2013.
Available from: http://www.jove.com/video/50217.
14. Piperdi B, Merla A, Perez-Soler R. Targeting angiogenesis in squamous nonsmall cell lung cancer. Drugs 2014;74:403–13.
15. Das M, Wakelee H. Targeting VEGF in lung cancer. Expert Opin Ther Targets
2012;16:395–406.
www.aacrjournals.org
16. Coelho AL, Gomes MP, Catarino RJ, Rolfo C, Lopes AM, Medeiros RM, et al.
Angiogenesis in NSCLC: is vessel co-option the trunk that sustains the
branches? Oncotarget 2016 Feb 29. [Epub ahead of print].
17. Wagner EM, Mitzner W, Brown RH. Site of functional bronchopulmonary
anastomoses in sheep. Anat Rec 1999;254:360–6.
18. Ng QS, Goh V. Angiogenesis in non-small cell lung cancer: imaging
with perfusion computed tomography. J Thorac Imaging 2010;25:
142–50.
19. Fraioli F, Anzidei M, Zaccagna F, Mennini ML, Serra G, Gori B, et al. Wholetumor perfusion CT in patients with advanced lung adenocarcinoma
treated with conventional and antiangiogenetic chemotherapy: initial
experience. Radiology 2011;259:574–82.
20. Mitzner W, Lee W, Georgakopoulos D, Wagner E. Angiogenesis in the
mouse lung. Am J Pathol 2000;157:93–101.
21. Savai R, Wolf JC, Greschus S, Eul BG, Schermuly RT, Hanze J, et al. Analysis
of tumor vessel supply in Lewis lung carcinoma in mice by fluorescent
microsphere distribution and imaging with micro- and flat-panel computed tomography. Am J Pathol 2005;167:937–46.
22. Tan X, Carretero J, Chen Z, Zhang J, Wang Y, Chen J, et al. Loss of p53
attenuates the contribution of IL-6 deletion on suppressed tumor progression and extended survival in Kras-driven murine lung cancer. PLoS One
2013;8:e80885.
23. DuPage M, Dooley AL, Jacks T. Conditional mouse lung cancer models
using adenoviral or lentiviral delivery of Cre recombinase. Nat Protoc
2009;4:1064–72.
24. Kasinski AL, Slack FJ. miRNA-34 prevents cancer initiation and progression
in a therapeutically resistant K-ras and p53-induced mouse model of lung
adenocarcinoma. Cancer Res 2012;72:5576–87.
25. Zundelevich A, Elad-Sfadia G, Haklai R, Kloog Y. Suppression of lung
cancer tumor growth in a nude mouse model by the Ras inhibitor salirasib
(farnesylthiosalicylic acid). Mol Cancer Ther 2007;6:1765–73.
26. Tanaka T, Delong PA, Amin K, Henry A, Kruklitis R, Kapoor V, et al.
Treatment of lung cancer using clinically relevant oral doses of the cyclooxygenase-2 inhibitor rofecoxib: potential value as adjuvant therapy after
surgery. Ann Surg 2005;241:168–78.
27. Neyazaki T, Ikeda M, Seki Y, Egawa N, Suzuki C. Bronchial artery infusion
therapy for lung cancer. Cancer 1969;24:912–22.
Cancer Res; 76(20) October 15, 2016
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2016 American Association for Cancer
Research.
OF7
Published OnlineFirst August 28, 2016; DOI: 10.1158/0008-5472.CAN-16-1131
Eldridge et al.
28. Yoshida T, Kamada K, Miura K, Goto T, Ohshima S, Sato W, et al.
Successful treatment of hepatocellular carcinoma with lung metastasis
using hepatic and bronchial artery infusion chemotherapy. Intern Med
2014;53:2493–7.
29. Yan D, Zhou CW, Liu DZ, Chen Y, Zeng HY, Li H. [Evaluation of the
efficacy of bronchial arterial infusion chemotherapy for the treatment of
central non-small cell lung cancer]. Zhonghua Zhong Liu Za Zhi
2011;33:302–4.
30. Razazi K, Parrot A, Khalil A, Djibre M, Gounant V, Assouad J, et al. J. Severe
haemoptysis in patients with nonsmall cell lung carcinoma. Eur Respir J
2015;45:756–64.
31. Fujita T, Tanabe M, Moritani K, Matsunaga N, Matsumoto T. Immediate
and late outcomes of bronchial and systemic artery embolization for
OF8 Cancer Res; 76(20) October 15, 2016
palliative treatment of patients with nonsmall-cell lung cancer having
hemoptysis. Am J Hosp Palliat Care 2014;31:602–7.
32. Wu W, Onn A, Isobe T, Itasaka S, Langley RR, Shitani T, et al. Targeted
therapy of orthotopic human lung cancer by combined vascular endothelial growth factor and epidermal growth factor receptor signaling blockade.
Mol Cancer Ther 2007;6:471–83.
33. Yuan D, Xia M, Meng G, Xu C, Song Y, Wei J. Anti-angiogenic efficacy of 50 triphosphate siRNA combining VEGF silencing and RIG-I activation in
NSCLCs. Oncotarget 2015;6:29664–74.
34. Ito K, Semba T, Uenaka T, Wakabayashi T, Asada M, Funahashi Y. Enhanced
anti-angiogenic effect of E7820 in combination with erlotinib in epidermal
growth factor receptor-tyrosine kinase inhibitor-resistant non-small-cell
lung cancer xenograft models. Cancer Sci 2014;105:1023–31.
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2016 American Association for Cancer
Research.
Published OnlineFirst August 28, 2016; DOI: 10.1158/0008-5472.CAN-16-1131
Bronchial Artery Angiogenesis Drives Lung Tumor Growth
Lindsey Eldridge, Aigul Moldobaeva, Qiong Zhong, et al.
Cancer Res Published OnlineFirst August 28, 2016.
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