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Tumors exposed to acute cyclic hypoxia show increased vessel density and delayed blood supply
Microvascular Research 85 (2013) 10–15
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Tumors exposed to acute cyclic hypoxia show increased vessel density and delayed blood supply Jon-Vidar Gaustad ⁎, Trude Golimo Simonsen, Ana Maria Acosta Roa, Einar K. Rofstad Group of Radiation Biology and Tumor Physiology, Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway
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Article history: Accepted 4 November 2012 Available online 12 November 2012
a b s t r a c t The purpose of this study was to investigate the effect of acute cyclic hypoxia on tumor vasculature. A-07 human melanoma xenografts growing in dorsal window chambers were used as tumor model. Acute cyclic hypoxia was induced by periodically exposing tumor-bearing mice to a low oxygen atmosphere. The hypoxia treatment consisted of 12 cycles of 10 min of low O2 (8% O2 in N2) followed by 10 min of air for a total of 4 hr. The treatment started the first day after tumor initiation, and was given daily for 9 days. Vascular morphology was assessed from high-resolution transillumination images, and tumor blood supply was assessed from first-pass imaging movies recorded after a bolus of 155 kDa tetramethylrhodamine isothiocyanate-labeled dextran had been administered intravenously. Hypoxia-treated tumors showed increased vessel density, decreased interstitial distance, and delayed blood supply compared to control tumors. The increase in vessel density was attributed to an increased number of small vessels. In conclusion, acute cyclic hypoxia induced angiogenesis in A-07 tumors resulting in increased density of small-diameter vessels and delayed tumor blood supply. © 2012 Elsevier Inc. All rights reserved.
Introduction Most experimental and human tumors are heterogeneous in oxygen tension, and develop regions with hypoxic cells during growth (Höckel and Vaupel, 2001). Experimental studies have demonstrated that tumors with extensive hypoxic regions are resistant to several types of therapy, including radiation therapy, photodynamic therapy and some forms of chemotherapy (Brown, 1999; Höckel and Vaupel, 2001). Moreover, it has been shown that hypoxia may promote malignant progression and metastatic dissemination in experimental tumor models (Rofstad, 2000). Clinical studies have revealed that extensive hypoxia in the primary tumor is associated with locoregional treatment failure and poor disease-free and overall survival rates in several types of cancer including cervical carcinoma, head and neck cancer, and soft tissue sarcoma (Brizel et al., 1999; Nordsmark et al., 2001; Sundfør et al., 2000). Two main causes of tumor hypoxia have been recognized: permanent limitations in oxygen diffusion (Thomlinson and Gray, 1955) and transient limitations in blood perfusion (Chaplin et al., 1986). Permanent limitations in oxygen diffusion are a consequence of increased intervessel
Abbreviations: GFP, green fluorescence protein; TRITC, tetramethylrhodamine isothiocyanate; VEGF, vascular endothelial growth factor. ⁎ Corresponding author at: Group of Radiation Biology and Tumor Physiology, Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, N-0310 Oslo, Norway. Fax: +47 2278 1207. E-mail address: [email protected] (J.-V. Gaustad). 0026-2862/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.mvr.2012.11.002
distances, and give rise to hypoxic regions far from blood vessels. If left untreated, tumor cells in these regions are expected to remain hypoxic until they die because of a lack of oxygen and nutrients (Durand and Sham, 1998). Hypoxia caused by permanent limitations in oxygen diffusion is referred to as chronic hypoxia or diffusion-limited hypoxia. Transient limitations in blood perfusion can be caused by transient intravessel stasis or temporal variation in microvessel erythrocyte flux (Kimura et al., 1996). Tumor cells found downstream of perfusionimpairing vessel abnormalities are believed to experience several short-term periods of hypoxia during their lifetime (Dewhirst et al., 2008). Hypoxia caused by transient limitations in blood perfusion is referred to as acute hypoxia or perfusion-limited hypoxia. The impact of hypoxia on spontaneous metastasis has been studied by exposing tumor-bearing mice to a low O2 atmosphere. It has been shown that acute cyclic hypoxia increases lung micrometastasis in mice bearing KHT murine fibrosarcomas (Cairns et al., 2001) and lymph node metastasis in mice bearing ME180 human cervical carcinoma xenografts (Cairns and Hill, 2004). In KHT tumors, hypoxia treatment increased resistance to apoptosis by upregulating Mdm2 (murine double minute-2, an inhibitor of p53 transcriptional activation), causing increased survival of tumor cells arrested in the lungs (Zhang and Hill, 2004). In ME180 xenografts, several metastasisrelated genes were upregulated by the hypoxia treatment, including vascular endothelial growth factor-C (VEGF-C), chemokine receptor-4, urokinase-type plasminogen activator receptor, transformed 3T3 double minute-2, and osteopontin (Chaudary and Hill, 2009). Moreover, suppression of VEGF-C or VEGFR-3 was shown to inhibit the hypoxiainduced increase in lymph node metastasis, demonstrating that the
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VEGFC/VEGFR-3 signaling pathway was involved in hypoxia-induced metastasis in ME180 xenografts (Chaudary et al., 2011). In our laboratory, we have investigated the effect of naturally occurring hypoxia on spontaneous metastasis in human melanoma xenografts. We have shown that tumors that metastasize to lung or lymph nodes have higher fractions of both chronically and acutely hypoxic cells than tumors that do not metastasize, and we have demonstrated that acutely hypoxic tumor cells may have a greater metastatic potential than chronically hypoxic tumor cells (Rofstad et al., 2007). We have also studied the effect of hypoxia on spontaneous metastasis by exposing tumor-bearing mice to acute cyclic hypoxia. Acute cyclic hypoxia increased the incidence of lung micrometastases in mice bearing A-07 human melanoma xenografts (Rofstad et al., 2010). The hypoxia-treated A-07 tumors showed upregulation of VEGF-A, increased microvascular density, and increased blood perfusion. These observations suggested that the hypoxia treatment induced angiogenesis, facilitating intravasation and metastatic dissemination in A-07 tumors. Interestingly, the mechanisms for hypoxiainduced metastasis found in KHT murine fibrosarcomas and ME180 human cervical carcinoma xenografts did not involve increased angiogenesis (Cairns and Hill, 2004; Zhang and Hill, 2004). Taken together, these studies thus illustrate that acute cyclic hypoxia may promote metastasis by fundamentally different mechanisms in different tumor models. The aim of the current study was to investigate the effect of acute cyclic hypoxia on tumor vasculature in more detail. For this purpose, A-07 tumors growing in dorsal window chambers were exposed to acute cyclic hypoxia, and tumor vasculature was examined with intravital microscopy techniques. We report that A-07 tumors exposed to acute cyclic hypoxia show increased density of small-diameter vessels resulting in delayed
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tumor blood supply. To our knowledge, this is the first study evaluating the effect of hypoxia exposure in vivo in tumors growing in window chambers. Materials and methods Mice Adult (8–10 weeks of age) female BALB/c nu/nu mice were used as host animals for dorsal window chamber preparations. The mice were bred at our institute and maintained under specific pathogen-free conditions at constant temperature (24–26 °C) and humidity (30–50%). After implantation of dorsal window chambers, the mice were kept at a temperature of 32 °C and a humidity of 60–70%. The animal experiments were approved by the Institutional Committee on Research Animal Care and were done according to the Interdisciplinary Principles and Guidelines for the Use of Animals in Research, Marketing, and Education (New York Academy of Sciences, New York, NY). Cells and multicellular spheroids A-07 human melanoma cells (Rofstad, 1994) were constitutively transfected with green fluorescence protein (GFP) by lipofection. The transfected cells used in the present experiments were obtained from our frozen stock and were grown as monolayers in RPMI 1640 (25 mM HEPES and L-glutamine) supplemented with 13% bovine calf serum, 250 μg/mL penicillin, 50 μg/mL streptomycin, and 700 μg/mL genetecin. Multicellular spheroids were produced and maintained by using a liquid-overlay culture technique (Rofstad et al., 1986).
Fig. 1. Intravital microscopy images of a representative control A-07 tumor (A) and a representative hypoxia-treated A-07 tumor (B). The images were recorded 7 (left panels) and 10 days (right panels) after tumor initiation. Tumor area is delineated by a solid black line.
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Anesthesia Window chamber implantation and intravital microscopy examinations were carried out with anesthetized mice. Fentanyl citrate (Janssen Pharmaceutica, Beerse, Belgium), fluanisone (Janssen Pharmaceutica), and midazolam (Hoffmann-La Roche, Basel, Switzerland) were administered i.p. in doses of 0.63 mg/kg, 20 mg/kg, and 10 mg/kg, respectively. After surgery, the mice were given a single injection of buprenorphine (Temgesic; Schering-Plough, Brussels, Belgium) i.p. in a dose of 0.12 mg/kg to relieve pain. Window chamber preparations Window chambers were implanted into the dorsal skin fold as described previously (Brurberg et al., 2008). Briefly, the chamber consisted of two parallel frames, and after implantation, the frames sandwiched an extended double layer of skin. Before the chamber was implanted, a circular hole with a diameter of approximately 6.0 mm was made in one of the skin layers. A plastic window with a diameter of 6.0 mm was attached to the frame on the surgical side with a clip to provide visual access to the fascial side of the opposite skin layer. Tumors were initiated by implanting spheroids with a diameter of 200 to 400 μm onto the exposed skin layer. Hypoxia treatment Unanesthetized mice were placed in an in-house-made incubation chamber and exposed to a continuous flow of a humidified gas mixture at room temperature to induce hypoxia. The hypoxia treatment consisted
of 12 cycles of 10 min of 8% O2 in N2 followed by 10 min of air for a total of 4 hr. The hypoxia treatment began on the first day after tumor spheroid implantation and was given once per day for 9 succeeding days. Control mice were kept at room temperature and were allowed to breathe air during the daily 4 hr treatments.
Intravital microscopy The mice were kept in a specially constructed holder that fixed the window chamber to the microscope stage during intravital microscopy. The body core temperature was kept at 37 to 38 °C by using a hot-air generator. Imaging was performed by using an inverted fluorescence microscope equipped with filters for green and red light (IX-71; Olympus, Munich, Germany), a black and white CCD camera (C9300-024; Hamamatsu Photonics, Hamamatsu, Japan), and appropriate image acquisition software (Wasabi; Hamamatsu Photonics). Tumor vasculature was visualized by using transillumination and filters for green light, and tumor vascular networks were mapped by recording 1–4 single frames with a ×4 objective lens resulting in a field of view of 3.80× 3.80 mm2 and a pixel size of 3.7 × 3.7 μm2. To study vascular function, first-pass imaging movies were recorded after a 0.2 mL bolus of 50 mg/mL tetramethylrhodamine isothiocyanate-labeled dextran (Sigma-Aldrich, Schnelldorf, Germany) with a molecular weight of 155 kDa was injected into the lateral tail vein. First-pass imaging movies were recorded at a frame rate of 22.3 frames per second by using a × 2 objective lens, resulting in a time resolution of 44.8 ms, a field of view of 5.97 × 5.97 mm 2, and a pixel size of 7.5 × 7.5 μm 2. All recordings were stored and analyzed offline. Tumor size (i.e., tumor area) was calculated from the number of pixels showing GFP fluorescence.
Fig. 2. Vessel density (VD) of all vessels (A), VD of small vessels (B), VD of large vessels (C), interstitial distance (D), vessel segment length (E), and vessel tortuosity (F) in control A-07 tumors (○) and hypoxia-treated A-07 tumors (●) 7 and 10 days after tumor initiation. Small vessels refer to vessels with diameter b20 μm, whereas large vessels refer to vessels with diameter >20 μm. Points, individual tumors; horizontal lines, mean values.
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Analysis of vascular morphology
Acute cyclic hypoxia increased intratumor heterogeneity in vessel density
Two-dimensional projected vascular masks were produced manually from transillumination images recorded with the ×4 objective lens. Interstitial distance (i.e., the distance from a tumor pixel outside the vascular mask to the nearest pixel within the vascular mask) was computed from the vascular masks (Gaustad et al., 2008). Vessel density (i.e., total vessel length per mm2 tumor area) was calculated from skeletons of vascular masks (Gaustad et al., 2012). Vessel segment length and vessel tortuosity were calculated from ~ 50 randomly selected vessel segments. Vessel tortuosity (T) was defined as T=(SL− S)*100%/SL, where SL represents the segment length (i.e., the distance between the branching points along the vessel) and S represents the shortest distance between the branching points (i.e., the distance between the branching points along a straight line) (Gaustad et al., 2012).
To investigate intratumor heterogeneity in vessel density, tumors were divided into 3 concentric regions of interest (ROIs) as illustrated in Fig. 3A. Hypoxia-treated tumors showed significantly higher vessel density than control tumors in all ROIs (Fig. 3B). Moreover, significant differences in vessel density were not found between any of the ROIs in control tumors, whereas hypoxia-treated tumors showed significantly higher vessel density in the central and middle ROIs compared to the peripheral ROI (Fig. 3B). These observations suggest that the hypoxia treatment increased intratumor heterogeneity in vessel density.
Analysis of blood supply time Two-dimensional projected vascular masks were produced from the movies recorded with the ×2 objective lens as described previously (Gaustad et al., 2008). Blood supply time (BST) images were produced by assigning a BST value to each pixel of the vascular masks. The BST of a pixel was defined as the time difference between the frame showing maximum fluorescence intensity in the pixel and the frame showing maximum fluorescence intensity in the main tumor supplying artery, as described in detail previously (Øye et al., 2008). Median BST and BST range (i.e., the range of BST values in the BST frequency distribution with a frequency>10% of the maximum frequency) were calculated from BST frequency distributions.
Acute cyclic hypoxia increased BST To investigate the effect of acute cyclic hypoxia on blood supply, firstpass imaging movies were recorded and BST images and BST frequency distributions were produced. Treatment with acute cyclic hypoxia increased BST. This is illustrated qualitatively in Figs. 4A–B which shows the BST image and the corresponding BST frequency distribution of a representative control tumor (Fig. 4A) and a representative hypoxia-treated tumor (Fig. 4B). Quantitative studies showed that hypoxia-treated tumors had significantly higher median BST (Fig. 4C) and significantly increased range of BST-values (Fig. 4D) compared to control tumors.
Statistical analysis Statistical comparisons of data were carried out by the Student's t test when the data complied with the conditions of normality and equal variance. Under other conditions, comparisons were done by nonparametric analysis using the Mann–Whitney rank sum test. Probability values of Pb 0.05, determined from two-sided tests, were considered significant. The statistical analysis was performed by using the SigmaStat statistical software (SPSS Science, Chicago, IL, USA). Results Acute cyclic hypoxia increased vessel density To investigate the effect of acute cyclic hypoxia on tumor vasculature, hypoxia-treated tumors and untreated control tumors were subjected to intravital microscopy before treatment was initiated (day 0) and 3 times during the treatment period (day 4, 7 and 10). By day 7, both control and hypoxia-treated tumors were vascularised. Hypoxia-treated tumors showed increased vessel density compared to control tumors on day 7 and 10. This is illustrated qualitatively in Fig. 1 which shows intravital microscopy images of a representative control tumor (Fig. 1A) and a representative hypoxia-treated tumor (Fig. 1B). To quantify these qualitative observations, vascular masks were produced and vessel densities and interstitial distances were calculated (Figs. 2A–D). Hypoxia-treated tumors showed significantly higher vessel densities than control tumors (Fig. 2A). The density of small-diameter vessels was significantly increased in hypoxia-treated tumors (Fig. 2B), whereas the density of large-diameter vessels did not differ between untreated and hypoxia-treated tumors (Fig. 2C). In line with the increases in vessel density, hypoxia-treated tumors showed significantly lower interstitial distances than control tumors (Fig. 2D). Hypoxia-treated tumors also tended to show shorter vessel segment lengths than control tumors (Fig. 2E), but did not differ from control tumors in vessel tortuosity (Fig. 2F).
Fig. 3. A, intravital microscopy image of a representative hypoxia-treated A-07 tumor illustrating how tumors were divided into 3 concentric regions of interest (ROIs). The tumor ROIs are bounded by lines drawn at distances of n·R/3 from the tumor center, where R is tumor radius and n is ROI number (#1–#3). B, vessel density in different tumor ROIs in control A-07 tumors (○) and hypoxia-treated A-07 tumors (●) 10 days after tumor initiation. Points, individual tumors; horizontal lines, mean values.
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Fig. 4. A-B, blood supply time (BST) image and corresponding BST frequency distribution of a representative control A-07 tumor (A) and a representative hypoxia-treated A-07 tumor (B). Color bars, BST scale in seconds; solid vertical lines, median BST. C–D, median BST (C) and BST range (D) in control A-07 tumors (○) and hypoxia-treated A-07 tumors (●) 10 days after tumor initiation. Points, individual tumors; horizontal lines, mean values. BST range refers to the range of BST-values in the BST frequency distribution with a frequency> 10% of the maximum frequency. The BST range is indicated with dotted vertical lines in the BST frequency distributions (A–B).
Acute cyclic hypoxia did not increase tumor growth Hypoxia-treated tumors did not differ from control tumors in growth rate despite the observations that hypoxia treatment induced changes in vascular morphology and BST. This is shown in Fig. 5. Discussion A-07 human melanoma xenografts grown in dorsal window chambers were used as tumor model. We have previously demonstrated that tissue pO2 in A-07 tumors varies cyclically with time during acute cyclic hypoxia treatment, by using the OxyLite fiberoptic oxygen-sensing device (Rofstad et al., 2010). Tumor pO2 decreased rapidly to 0 mm Hg when tumor-bearing mice were exposed to the low O2 atmosphere (8% O2 in N2), and increased rapidly to the pretreatment level when the mice were exposed to normal air. Moreover, we have shown that small A-07 tumors do not contain naturally occurring hypoxia (Rofstad et al., 2002). Consequently, effects of artificially imposed acute cyclic hypoxia could be investigated in this tumor line without having to consider confounding effects from naturally occurring hypoxia. Tumors growing in dorsal window chambers are particularly wellsuited for evaluating treatment-induced effects on tumor vasculature, because both the morphology and function of tumor vasculature can be studied repetitively by using intravital microscopy techniques (Jain et al., 2002; Tozer et al., 2005). In the present study, vascular morphology was assessed by mapping tumor vascular networks with high-resolution transillumination images and filters for green light. In these images, only vessels with erythrocytes are visible and, consequently, the morphological analysis was based on vessels with erythrocytes as opposed to dysfunctional vessels with plasma only.
Vascular function was assessed by measuring BST. We have previously shown that the BST-assay is highly reproducible and sufficiently sensitive to detect gradients in BST along vessel segments. We have also shown that the spatial resolution used in this assay is sufficient to identify the majority of tumor vessels (Brurberg et al., 2008; Gaustad et al., 2009; Øye et al., 2008). We show that acute cyclic hypoxia increased vessel density in A-07 human melanoma xenografts. This observation is consistent with our previous work with intradermal flank tumors, where
Fig. 5. Tumor size in control A-07 tumors (○) and hypoxia-treated A-07 tumors (●) versus time. Points, average of 8 tumors; bars, SE.
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increased microvascular density, as assessed by CD-31 staining, and increased perfusion, as assessed by dynamic contrast-enhanced magnetic resonance imaging, were observed in A-07 tumors exposed to acute cyclic hypoxia (Rofstad et al., 2010). In the present study, we also show that the hypoxia-induced increase in vessel density was caused by an increased number of small vessels, and that the hypoxia treatment increased the intratumor heterogeneity in vessel density. The hypoxia-induced angiogenesis was probably caused by an upregulation of VEGF-A. This suggestion is based on our previous findings that acute cyclic hypoxia increased the expression of VEGF-A in A-07 tumors, and that treatment with anti-VEGF-A antibody reduced microvascular density and the number of metastases (Rofstad et al., 2010). In the same study, histological preparations of hypoxiatreated tumors did not show increased staining for interleukin-8, platelet-derived endothelial cell growth factor, or basic fibroblast growth factor (Rofstad et al., 2010). Under normoxic conditions, all these proangiogenic factors have been shown to mediate angiogenesis in A-07 tumors (Rofstad and Halsør, 2000). These data do not exclude the possibility that other pro- or antiangiogenic factors may be involved in hypoxia-induced angiogenesis in A-07 tumors. Malignant tumors show aberrant angiogenesis and, as a consequence, tumor vessels may display several structural and functional abnormalities (Vaupel et al., 1989). These abnormalities may increase the geometric resistance to blood flow, increase temporal fluctuations in blood flow, and reduce blood supply (Dewhirst et al., 1996; Jain, 1988). In the present study, we show that acute cyclic hypoxia increased BST. This observation implies that hypoxia-induced angiogenesis resulted in delayed tumor blood supply. The increase in BST was probably a consequence of increased geometric resistance to blood flow because hypoxia-treated tumors showed an increased number of small vessels (i.e. vessels with low diameter). Geometric resistance to blood flow in a single vessel is inversely correlated to vessel diameter to the forth power (Jain, 1988). Acute cyclic hypoxia did not induce angiogenesis in ME180 human cervical carcinoma xenografts or in PyMT transgenic breast cancer tumors (Cairns and Hill, 2004; Kalliomaki et al., 2008). Moreover, acute or chronic hypoxia did not induce angiogenesis in DS sarcomas (Thews et al., 2004). In these tumor models, the various hypoxia exposures decreased or did not alter the microvascular density, and the hypoxia exposures reduced tumor growth. The current study and these previously reported studies suggest that exposure to hypoxia in vivo may induce angiogenesis in some but not all tumor models. Hypoxia-induced metastasis may thus be attributed to increased angiogenic activity in some tumor models and to angiogenesis-independent mechanisms in other tumor models. In summary, acute cyclic hypoxia induced angiogenesis in A-07 human melanoma xenografts. The hypoxia treatment increased vessel density by increasing the number of small vessels, and the increased number of small vessels delayed tumor blood supply.
Role of the funding source This work was supported by research funding from the Norwegian Cancer Society. The Norwegian Cancer Society had no involvement in study design, in the collection, analysis, and interpretation of data, in the writing of the report, or in the decision to submit the article for publication.
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Tumors exposed to acute cyclic hypoxia show increased vessel density and delayed blood supply Jon-Vidar Gaustad ⁎, Trude Golimo Simonsen, Ana Maria Acosta Roa, Einar K. Rofstad Group of Radiation Biology and Tumor Physiology, Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway
a r t i c l e
i n f o
Article history: Accepted 4 November 2012 Available online 12 November 2012
a b s t r a c t The purpose of this study was to investigate the effect of acute cyclic hypoxia on tumor vasculature. A-07 human melanoma xenografts growing in dorsal window chambers were used as tumor model. Acute cyclic hypoxia was induced by periodically exposing tumor-bearing mice to a low oxygen atmosphere. The hypoxia treatment consisted of 12 cycles of 10 min of low O2 (8% O2 in N2) followed by 10 min of air for a total of 4 hr. The treatment started the first day after tumor initiation, and was given daily for 9 days. Vascular morphology was assessed from high-resolution transillumination images, and tumor blood supply was assessed from first-pass imaging movies recorded after a bolus of 155 kDa tetramethylrhodamine isothiocyanate-labeled dextran had been administered intravenously. Hypoxia-treated tumors showed increased vessel density, decreased interstitial distance, and delayed blood supply compared to control tumors. The increase in vessel density was attributed to an increased number of small vessels. In conclusion, acute cyclic hypoxia induced angiogenesis in A-07 tumors resulting in increased density of small-diameter vessels and delayed tumor blood supply. © 2012 Elsevier Inc. All rights reserved.
Introduction Most experimental and human tumors are heterogeneous in oxygen tension, and develop regions with hypoxic cells during growth (Höckel and Vaupel, 2001). Experimental studies have demonstrated that tumors with extensive hypoxic regions are resistant to several types of therapy, including radiation therapy, photodynamic therapy and some forms of chemotherapy (Brown, 1999; Höckel and Vaupel, 2001). Moreover, it has been shown that hypoxia may promote malignant progression and metastatic dissemination in experimental tumor models (Rofstad, 2000). Clinical studies have revealed that extensive hypoxia in the primary tumor is associated with locoregional treatment failure and poor disease-free and overall survival rates in several types of cancer including cervical carcinoma, head and neck cancer, and soft tissue sarcoma (Brizel et al., 1999; Nordsmark et al., 2001; Sundfør et al., 2000). Two main causes of tumor hypoxia have been recognized: permanent limitations in oxygen diffusion (Thomlinson and Gray, 1955) and transient limitations in blood perfusion (Chaplin et al., 1986). Permanent limitations in oxygen diffusion are a consequence of increased intervessel
Abbreviations: GFP, green fluorescence protein; TRITC, tetramethylrhodamine isothiocyanate; VEGF, vascular endothelial growth factor. ⁎ Corresponding author at: Group of Radiation Biology and Tumor Physiology, Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, N-0310 Oslo, Norway. Fax: +47 2278 1207. E-mail address: [email protected] (J.-V. Gaustad). 0026-2862/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.mvr.2012.11.002
distances, and give rise to hypoxic regions far from blood vessels. If left untreated, tumor cells in these regions are expected to remain hypoxic until they die because of a lack of oxygen and nutrients (Durand and Sham, 1998). Hypoxia caused by permanent limitations in oxygen diffusion is referred to as chronic hypoxia or diffusion-limited hypoxia. Transient limitations in blood perfusion can be caused by transient intravessel stasis or temporal variation in microvessel erythrocyte flux (Kimura et al., 1996). Tumor cells found downstream of perfusionimpairing vessel abnormalities are believed to experience several short-term periods of hypoxia during their lifetime (Dewhirst et al., 2008). Hypoxia caused by transient limitations in blood perfusion is referred to as acute hypoxia or perfusion-limited hypoxia. The impact of hypoxia on spontaneous metastasis has been studied by exposing tumor-bearing mice to a low O2 atmosphere. It has been shown that acute cyclic hypoxia increases lung micrometastasis in mice bearing KHT murine fibrosarcomas (Cairns et al., 2001) and lymph node metastasis in mice bearing ME180 human cervical carcinoma xenografts (Cairns and Hill, 2004). In KHT tumors, hypoxia treatment increased resistance to apoptosis by upregulating Mdm2 (murine double minute-2, an inhibitor of p53 transcriptional activation), causing increased survival of tumor cells arrested in the lungs (Zhang and Hill, 2004). In ME180 xenografts, several metastasisrelated genes were upregulated by the hypoxia treatment, including vascular endothelial growth factor-C (VEGF-C), chemokine receptor-4, urokinase-type plasminogen activator receptor, transformed 3T3 double minute-2, and osteopontin (Chaudary and Hill, 2009). Moreover, suppression of VEGF-C or VEGFR-3 was shown to inhibit the hypoxiainduced increase in lymph node metastasis, demonstrating that the
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VEGFC/VEGFR-3 signaling pathway was involved in hypoxia-induced metastasis in ME180 xenografts (Chaudary et al., 2011). In our laboratory, we have investigated the effect of naturally occurring hypoxia on spontaneous metastasis in human melanoma xenografts. We have shown that tumors that metastasize to lung or lymph nodes have higher fractions of both chronically and acutely hypoxic cells than tumors that do not metastasize, and we have demonstrated that acutely hypoxic tumor cells may have a greater metastatic potential than chronically hypoxic tumor cells (Rofstad et al., 2007). We have also studied the effect of hypoxia on spontaneous metastasis by exposing tumor-bearing mice to acute cyclic hypoxia. Acute cyclic hypoxia increased the incidence of lung micrometastases in mice bearing A-07 human melanoma xenografts (Rofstad et al., 2010). The hypoxia-treated A-07 tumors showed upregulation of VEGF-A, increased microvascular density, and increased blood perfusion. These observations suggested that the hypoxia treatment induced angiogenesis, facilitating intravasation and metastatic dissemination in A-07 tumors. Interestingly, the mechanisms for hypoxiainduced metastasis found in KHT murine fibrosarcomas and ME180 human cervical carcinoma xenografts did not involve increased angiogenesis (Cairns and Hill, 2004; Zhang and Hill, 2004). Taken together, these studies thus illustrate that acute cyclic hypoxia may promote metastasis by fundamentally different mechanisms in different tumor models. The aim of the current study was to investigate the effect of acute cyclic hypoxia on tumor vasculature in more detail. For this purpose, A-07 tumors growing in dorsal window chambers were exposed to acute cyclic hypoxia, and tumor vasculature was examined with intravital microscopy techniques. We report that A-07 tumors exposed to acute cyclic hypoxia show increased density of small-diameter vessels resulting in delayed
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tumor blood supply. To our knowledge, this is the first study evaluating the effect of hypoxia exposure in vivo in tumors growing in window chambers. Materials and methods Mice Adult (8–10 weeks of age) female BALB/c nu/nu mice were used as host animals for dorsal window chamber preparations. The mice were bred at our institute and maintained under specific pathogen-free conditions at constant temperature (24–26 °C) and humidity (30–50%). After implantation of dorsal window chambers, the mice were kept at a temperature of 32 °C and a humidity of 60–70%. The animal experiments were approved by the Institutional Committee on Research Animal Care and were done according to the Interdisciplinary Principles and Guidelines for the Use of Animals in Research, Marketing, and Education (New York Academy of Sciences, New York, NY). Cells and multicellular spheroids A-07 human melanoma cells (Rofstad, 1994) were constitutively transfected with green fluorescence protein (GFP) by lipofection. The transfected cells used in the present experiments were obtained from our frozen stock and were grown as monolayers in RPMI 1640 (25 mM HEPES and L-glutamine) supplemented with 13% bovine calf serum, 250 μg/mL penicillin, 50 μg/mL streptomycin, and 700 μg/mL genetecin. Multicellular spheroids were produced and maintained by using a liquid-overlay culture technique (Rofstad et al., 1986).
Fig. 1. Intravital microscopy images of a representative control A-07 tumor (A) and a representative hypoxia-treated A-07 tumor (B). The images were recorded 7 (left panels) and 10 days (right panels) after tumor initiation. Tumor area is delineated by a solid black line.
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Anesthesia Window chamber implantation and intravital microscopy examinations were carried out with anesthetized mice. Fentanyl citrate (Janssen Pharmaceutica, Beerse, Belgium), fluanisone (Janssen Pharmaceutica), and midazolam (Hoffmann-La Roche, Basel, Switzerland) were administered i.p. in doses of 0.63 mg/kg, 20 mg/kg, and 10 mg/kg, respectively. After surgery, the mice were given a single injection of buprenorphine (Temgesic; Schering-Plough, Brussels, Belgium) i.p. in a dose of 0.12 mg/kg to relieve pain. Window chamber preparations Window chambers were implanted into the dorsal skin fold as described previously (Brurberg et al., 2008). Briefly, the chamber consisted of two parallel frames, and after implantation, the frames sandwiched an extended double layer of skin. Before the chamber was implanted, a circular hole with a diameter of approximately 6.0 mm was made in one of the skin layers. A plastic window with a diameter of 6.0 mm was attached to the frame on the surgical side with a clip to provide visual access to the fascial side of the opposite skin layer. Tumors were initiated by implanting spheroids with a diameter of 200 to 400 μm onto the exposed skin layer. Hypoxia treatment Unanesthetized mice were placed in an in-house-made incubation chamber and exposed to a continuous flow of a humidified gas mixture at room temperature to induce hypoxia. The hypoxia treatment consisted
of 12 cycles of 10 min of 8% O2 in N2 followed by 10 min of air for a total of 4 hr. The hypoxia treatment began on the first day after tumor spheroid implantation and was given once per day for 9 succeeding days. Control mice were kept at room temperature and were allowed to breathe air during the daily 4 hr treatments.
Intravital microscopy The mice were kept in a specially constructed holder that fixed the window chamber to the microscope stage during intravital microscopy. The body core temperature was kept at 37 to 38 °C by using a hot-air generator. Imaging was performed by using an inverted fluorescence microscope equipped with filters for green and red light (IX-71; Olympus, Munich, Germany), a black and white CCD camera (C9300-024; Hamamatsu Photonics, Hamamatsu, Japan), and appropriate image acquisition software (Wasabi; Hamamatsu Photonics). Tumor vasculature was visualized by using transillumination and filters for green light, and tumor vascular networks were mapped by recording 1–4 single frames with a ×4 objective lens resulting in a field of view of 3.80× 3.80 mm2 and a pixel size of 3.7 × 3.7 μm2. To study vascular function, first-pass imaging movies were recorded after a 0.2 mL bolus of 50 mg/mL tetramethylrhodamine isothiocyanate-labeled dextran (Sigma-Aldrich, Schnelldorf, Germany) with a molecular weight of 155 kDa was injected into the lateral tail vein. First-pass imaging movies were recorded at a frame rate of 22.3 frames per second by using a × 2 objective lens, resulting in a time resolution of 44.8 ms, a field of view of 5.97 × 5.97 mm 2, and a pixel size of 7.5 × 7.5 μm 2. All recordings were stored and analyzed offline. Tumor size (i.e., tumor area) was calculated from the number of pixels showing GFP fluorescence.
Fig. 2. Vessel density (VD) of all vessels (A), VD of small vessels (B), VD of large vessels (C), interstitial distance (D), vessel segment length (E), and vessel tortuosity (F) in control A-07 tumors (○) and hypoxia-treated A-07 tumors (●) 7 and 10 days after tumor initiation. Small vessels refer to vessels with diameter b20 μm, whereas large vessels refer to vessels with diameter >20 μm. Points, individual tumors; horizontal lines, mean values.
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Analysis of vascular morphology
Acute cyclic hypoxia increased intratumor heterogeneity in vessel density
Two-dimensional projected vascular masks were produced manually from transillumination images recorded with the ×4 objective lens. Interstitial distance (i.e., the distance from a tumor pixel outside the vascular mask to the nearest pixel within the vascular mask) was computed from the vascular masks (Gaustad et al., 2008). Vessel density (i.e., total vessel length per mm2 tumor area) was calculated from skeletons of vascular masks (Gaustad et al., 2012). Vessel segment length and vessel tortuosity were calculated from ~ 50 randomly selected vessel segments. Vessel tortuosity (T) was defined as T=(SL− S)*100%/SL, where SL represents the segment length (i.e., the distance between the branching points along the vessel) and S represents the shortest distance between the branching points (i.e., the distance between the branching points along a straight line) (Gaustad et al., 2012).
To investigate intratumor heterogeneity in vessel density, tumors were divided into 3 concentric regions of interest (ROIs) as illustrated in Fig. 3A. Hypoxia-treated tumors showed significantly higher vessel density than control tumors in all ROIs (Fig. 3B). Moreover, significant differences in vessel density were not found between any of the ROIs in control tumors, whereas hypoxia-treated tumors showed significantly higher vessel density in the central and middle ROIs compared to the peripheral ROI (Fig. 3B). These observations suggest that the hypoxia treatment increased intratumor heterogeneity in vessel density.
Analysis of blood supply time Two-dimensional projected vascular masks were produced from the movies recorded with the ×2 objective lens as described previously (Gaustad et al., 2008). Blood supply time (BST) images were produced by assigning a BST value to each pixel of the vascular masks. The BST of a pixel was defined as the time difference between the frame showing maximum fluorescence intensity in the pixel and the frame showing maximum fluorescence intensity in the main tumor supplying artery, as described in detail previously (Øye et al., 2008). Median BST and BST range (i.e., the range of BST values in the BST frequency distribution with a frequency>10% of the maximum frequency) were calculated from BST frequency distributions.
Acute cyclic hypoxia increased BST To investigate the effect of acute cyclic hypoxia on blood supply, firstpass imaging movies were recorded and BST images and BST frequency distributions were produced. Treatment with acute cyclic hypoxia increased BST. This is illustrated qualitatively in Figs. 4A–B which shows the BST image and the corresponding BST frequency distribution of a representative control tumor (Fig. 4A) and a representative hypoxia-treated tumor (Fig. 4B). Quantitative studies showed that hypoxia-treated tumors had significantly higher median BST (Fig. 4C) and significantly increased range of BST-values (Fig. 4D) compared to control tumors.
Statistical analysis Statistical comparisons of data were carried out by the Student's t test when the data complied with the conditions of normality and equal variance. Under other conditions, comparisons were done by nonparametric analysis using the Mann–Whitney rank sum test. Probability values of Pb 0.05, determined from two-sided tests, were considered significant. The statistical analysis was performed by using the SigmaStat statistical software (SPSS Science, Chicago, IL, USA). Results Acute cyclic hypoxia increased vessel density To investigate the effect of acute cyclic hypoxia on tumor vasculature, hypoxia-treated tumors and untreated control tumors were subjected to intravital microscopy before treatment was initiated (day 0) and 3 times during the treatment period (day 4, 7 and 10). By day 7, both control and hypoxia-treated tumors were vascularised. Hypoxia-treated tumors showed increased vessel density compared to control tumors on day 7 and 10. This is illustrated qualitatively in Fig. 1 which shows intravital microscopy images of a representative control tumor (Fig. 1A) and a representative hypoxia-treated tumor (Fig. 1B). To quantify these qualitative observations, vascular masks were produced and vessel densities and interstitial distances were calculated (Figs. 2A–D). Hypoxia-treated tumors showed significantly higher vessel densities than control tumors (Fig. 2A). The density of small-diameter vessels was significantly increased in hypoxia-treated tumors (Fig. 2B), whereas the density of large-diameter vessels did not differ between untreated and hypoxia-treated tumors (Fig. 2C). In line with the increases in vessel density, hypoxia-treated tumors showed significantly lower interstitial distances than control tumors (Fig. 2D). Hypoxia-treated tumors also tended to show shorter vessel segment lengths than control tumors (Fig. 2E), but did not differ from control tumors in vessel tortuosity (Fig. 2F).
Fig. 3. A, intravital microscopy image of a representative hypoxia-treated A-07 tumor illustrating how tumors were divided into 3 concentric regions of interest (ROIs). The tumor ROIs are bounded by lines drawn at distances of n·R/3 from the tumor center, where R is tumor radius and n is ROI number (#1–#3). B, vessel density in different tumor ROIs in control A-07 tumors (○) and hypoxia-treated A-07 tumors (●) 10 days after tumor initiation. Points, individual tumors; horizontal lines, mean values.
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Fig. 4. A-B, blood supply time (BST) image and corresponding BST frequency distribution of a representative control A-07 tumor (A) and a representative hypoxia-treated A-07 tumor (B). Color bars, BST scale in seconds; solid vertical lines, median BST. C–D, median BST (C) and BST range (D) in control A-07 tumors (○) and hypoxia-treated A-07 tumors (●) 10 days after tumor initiation. Points, individual tumors; horizontal lines, mean values. BST range refers to the range of BST-values in the BST frequency distribution with a frequency> 10% of the maximum frequency. The BST range is indicated with dotted vertical lines in the BST frequency distributions (A–B).
Acute cyclic hypoxia did not increase tumor growth Hypoxia-treated tumors did not differ from control tumors in growth rate despite the observations that hypoxia treatment induced changes in vascular morphology and BST. This is shown in Fig. 5. Discussion A-07 human melanoma xenografts grown in dorsal window chambers were used as tumor model. We have previously demonstrated that tissue pO2 in A-07 tumors varies cyclically with time during acute cyclic hypoxia treatment, by using the OxyLite fiberoptic oxygen-sensing device (Rofstad et al., 2010). Tumor pO2 decreased rapidly to 0 mm Hg when tumor-bearing mice were exposed to the low O2 atmosphere (8% O2 in N2), and increased rapidly to the pretreatment level when the mice were exposed to normal air. Moreover, we have shown that small A-07 tumors do not contain naturally occurring hypoxia (Rofstad et al., 2002). Consequently, effects of artificially imposed acute cyclic hypoxia could be investigated in this tumor line without having to consider confounding effects from naturally occurring hypoxia. Tumors growing in dorsal window chambers are particularly wellsuited for evaluating treatment-induced effects on tumor vasculature, because both the morphology and function of tumor vasculature can be studied repetitively by using intravital microscopy techniques (Jain et al., 2002; Tozer et al., 2005). In the present study, vascular morphology was assessed by mapping tumor vascular networks with high-resolution transillumination images and filters for green light. In these images, only vessels with erythrocytes are visible and, consequently, the morphological analysis was based on vessels with erythrocytes as opposed to dysfunctional vessels with plasma only.
Vascular function was assessed by measuring BST. We have previously shown that the BST-assay is highly reproducible and sufficiently sensitive to detect gradients in BST along vessel segments. We have also shown that the spatial resolution used in this assay is sufficient to identify the majority of tumor vessels (Brurberg et al., 2008; Gaustad et al., 2009; Øye et al., 2008). We show that acute cyclic hypoxia increased vessel density in A-07 human melanoma xenografts. This observation is consistent with our previous work with intradermal flank tumors, where
Fig. 5. Tumor size in control A-07 tumors (○) and hypoxia-treated A-07 tumors (●) versus time. Points, average of 8 tumors; bars, SE.
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increased microvascular density, as assessed by CD-31 staining, and increased perfusion, as assessed by dynamic contrast-enhanced magnetic resonance imaging, were observed in A-07 tumors exposed to acute cyclic hypoxia (Rofstad et al., 2010). In the present study, we also show that the hypoxia-induced increase in vessel density was caused by an increased number of small vessels, and that the hypoxia treatment increased the intratumor heterogeneity in vessel density. The hypoxia-induced angiogenesis was probably caused by an upregulation of VEGF-A. This suggestion is based on our previous findings that acute cyclic hypoxia increased the expression of VEGF-A in A-07 tumors, and that treatment with anti-VEGF-A antibody reduced microvascular density and the number of metastases (Rofstad et al., 2010). In the same study, histological preparations of hypoxiatreated tumors did not show increased staining for interleukin-8, platelet-derived endothelial cell growth factor, or basic fibroblast growth factor (Rofstad et al., 2010). Under normoxic conditions, all these proangiogenic factors have been shown to mediate angiogenesis in A-07 tumors (Rofstad and Halsør, 2000). These data do not exclude the possibility that other pro- or antiangiogenic factors may be involved in hypoxia-induced angiogenesis in A-07 tumors. Malignant tumors show aberrant angiogenesis and, as a consequence, tumor vessels may display several structural and functional abnormalities (Vaupel et al., 1989). These abnormalities may increase the geometric resistance to blood flow, increase temporal fluctuations in blood flow, and reduce blood supply (Dewhirst et al., 1996; Jain, 1988). In the present study, we show that acute cyclic hypoxia increased BST. This observation implies that hypoxia-induced angiogenesis resulted in delayed tumor blood supply. The increase in BST was probably a consequence of increased geometric resistance to blood flow because hypoxia-treated tumors showed an increased number of small vessels (i.e. vessels with low diameter). Geometric resistance to blood flow in a single vessel is inversely correlated to vessel diameter to the forth power (Jain, 1988). Acute cyclic hypoxia did not induce angiogenesis in ME180 human cervical carcinoma xenografts or in PyMT transgenic breast cancer tumors (Cairns and Hill, 2004; Kalliomaki et al., 2008). Moreover, acute or chronic hypoxia did not induce angiogenesis in DS sarcomas (Thews et al., 2004). In these tumor models, the various hypoxia exposures decreased or did not alter the microvascular density, and the hypoxia exposures reduced tumor growth. The current study and these previously reported studies suggest that exposure to hypoxia in vivo may induce angiogenesis in some but not all tumor models. Hypoxia-induced metastasis may thus be attributed to increased angiogenic activity in some tumor models and to angiogenesis-independent mechanisms in other tumor models. In summary, acute cyclic hypoxia induced angiogenesis in A-07 human melanoma xenografts. The hypoxia treatment increased vessel density by increasing the number of small vessels, and the increased number of small vessels delayed tumor blood supply.
Role of the funding source This work was supported by research funding from the Norwegian Cancer Society. The Norwegian Cancer Society had no involvement in study design, in the collection, analysis, and interpretation of data, in the writing of the report, or in the decision to submit the article for publication.
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