Disparate effects of endostatin on tumor vascular perfusion and hypoxia in two murine mammary carcinomas

PURPOSE: Recent results in the literature have demonstrated that the antiangiogenic agent endostatin can enhance antitumor effects when administered before or during radiotherapy. To better understand the underlying pathophysiologic basis for this radiosensitization, the current study investigated whether short-term endostatin administration is linked to alterations in tumor vascular perfusion and oxygen delivery. METHODS AND MATERIALS: Three daily doses of recombinant endostatin (20 mg/kg) were administered to two murine mammary carcinomas, the highly vascularized MCa-35 and the less vascularized MCa-4. Image analysis techniques were used to quantify (1) total and perfused vascular spacing, and (2) changes in tumor hypoxia as a function of distance from the nearest blood vessel. RESULTS: In MCa-35 tumors, endostatin had no effect on vessel spacing, tumor hypoxia, or tumor growth. In MCa-4 tumors, total and perfused vessel spacings were also unchanged, but tumor growth was inhibited, and tumor hypoxia significantly decreased. These tumors demonstrated an increased vascular functionality suggestive of an increase in the number of intermittently perfused vessels, without corresponding alterations in tumor oxygen consumption rate. CONCLUSIONS: Poorly vascularized, hypoxic mammary carcinomas were much more responsive to short-term endostatin treatment than well-vascularized, more homogeneously oxygenated tumors. Oxygen levels in the responsive tumors were transiently improved after treatment, which could have substantial implications with respect to the therapeutic effectiveness of combining antiangiogenic agents with conventional therapies.     

Influence of hydralazine administration on oxygenation in spontaneous and transplanted tumor models

PURPOSE: To examine the effects of hydralazine on vascular perfusion and hypoxia in spontaneous vs. first generation and long-term transplanted murine tumor models. METHODS AND MATERIALS: Total anatomic blood vessels were quantified using image analysis of CD31 stained frozen sections, perfused vessels by i.v. injection of fluorescent DiOC(7), and tumor hypoxia was measured using the EF5 hypoxia marker. KHT sarcomas, spontaneous mammary carcinomas, and first generation transplants of the spontaneous tumors were evaluated before and after i.p. administration of 5 mg/kg hydralazine. RESULTS: Although anatomic and perfused vessel spacings were similar among untreated tumors, response to hydralazine varied widely among the three tumor models. In KHT tumors, perfused vessel numbers decreased significantly at 30 min post-hydralazine, then recovered somewhat by 60 min. First-generation transplants showed a less substantial decrease in perfused vessels following hydralazine, which tapered off slightly by 60 min. Finally, spontaneous tumors had only a modest decrease in perfused vessel numbers, with complete recovery at 60 min. Although response of individual tumors varied widely, overall hypoxic marker uptake was significantly increased in both KHT and first generation tumors, and slightly reduced in the spontaneous tumors. CONCLUSION: Response to hydralazine varies substantially between transplanted and spontaneous tumor models. Results suggest that increased tumor pressure may be a critical factor in tumor response to hydralazine, possibly explaining tumor volume dependent variations.     

Spatial relationship between hypoxia and the (perfused) vascular network in a human glioma xenograft: a quantitative multi-parameter analysis

PURPOSE: To quantitatively study the spatial distribution of tumor hypoxia in relation to the perfused vasculature. METHODS AND MATERIALS: Using a human glioma xenograft model, nude mice were administered two different hypoxia markers (NITP or pimonidazole) and the perfusion marker Hoechst 33342. Frozen tumor sections were sequentially scanned for perfusion, hypoxia, and vasculature, respectively, to quantitate perfusion, vasculature, and hypoxia parameters in the same section. RESULTS: All tumors showed incomplete perfusion. Both NITP and pimonidazole stained the same hypoxic tumor areas. No statistically significant differences between the two markers were observed. The density of the perfused vessels was inversely related to the hypoxic fraction. At critical distances from perfused vessels, hypoxia occurred. These data suggest that predominantly diffusion-limited hypoxia was detected, based on the spatial distribution of nearby vessels. Also, the proportion of hypoxia distributed over arbitrary zones of 50 microm around perfused vessels was calculated. The largest proportion of hypoxia was found at distances beyond 100 microm from perfused vessels. CONCLUSION: With the multiple staining and functional microscopic imaging technique described here, the spatial relationship between perfused vessels and hypoxia was quantified in whole tumor cross-sections. The usefulness of this histologically-based method to quantitate morphological and physiological aspects of the tumor microenvironment was evaluated.     

Effect of VEGF receptor-2 antibody on vascular function and oxygenation in spontaneous and transplanted tumors.

BACKGROUND AND PURPOSE: The primary objectives of this study were to address two major questions. (1) Does VEGF receptor-2 antibody (DC101) produce detrimental effects on tumor vascular function and oxygenation that could compromise adjuvant therapies? (2) Is pathophysiological response to such antiangiogenic strategies different in transplanted versus primary spontaneous tumors? MATERIALS AND METHODS: The effects of early and late initiation DC101 treatment were evaluated using spontaneous murine mammary carcinomas and two markedly different transplanted mammary tumors, MCa-35 and MCa-4. Mice were administered DC101 or saline, tumors were frozen, and immunohistochemical staining was quantified using image analysis of multiply-stained frozen sections. Total blood vessels were identified using antibodies to CD31 or panendothelial antigen, perfused vessels via i.v. injection of fluorescent DiOC7, and tumor hypoxia by hypoxia marker (EF5) uptake. RESULTS: Tumor growth was significantly inhibited following DC101 administration in all tumor models. In general, early initiation DC101 treatment reduced perfused vessel counts and increased tumor hypoxia, while late initiation treatment had no significant impact on either. Results indicate that DC101 slows tumor growth through a decrease in vascular function, leading to increased tumor cell apoptosis and necrosis at sites distant from perfused blood vessels, and suggest that DC101 accelerates the rate at which tumor cells outgrow their functional vascular supply. CONCLUSIONS: Although highly variable among individual spontaneous tumors, the overall effects of DC101 on tumor hypoxia were quite similar between spontaneous and transplanted tumors. Since reductions in tumor oxygenation due to antiangiogenic treatment were transient, initial pathophysiological deficiencies that could compromise conventional therapies over the short-term may be of less relevance when administered over more extended treatment schedules.     

Microenvironmental adaptation of experimental tumours to chronic vs acute hypoxia

This study investigated long-term microenvironmental responses (oxygenation, perfusion, metabolic status, proliferation, vascular endothelial growth factor (VEGF) expression and vascularisation) to chronic hypoxia in experimental tumours. Experiments were performed using s.c.-implanted DS-sarcomas in rats. In order to induce more pronounced tumour hypoxia, one group of animals was housed in a hypoxic atmosphere (8% O(2)) for the whole period of tumour growth (chronic hypoxia). A second group was acutely exposed to inspiratory hypoxia for only 20 min prior to the measurements (acute hypoxia), whereas animals housed under normal atmospheric conditions served as controls. Acute hypoxia reduced the median oxygen partial pressure (pO(2)) dramatically (1 vs 10 mmHg in controls), whereas in chronically hypoxic tumours the pO(2) was significantly improved (median pO(2)=4 mmHg), however not reaching the control level. These findings reflect the changes in tumour perfusion where acutely hypoxic tumours show a dramatic reduction of perfused tumour vessels (maybe the result of a simultaneous reduction in arterial blood pressure). In animals under chronic inspiratory hypoxia, the number of perfused vessels increased (compared to acute hypoxia), although the perfusion pattern found in control tumours was not reached. In the chronically hypoxic animals, tumour cell proliferation and tumour growth were significantly reduced, whereas no differences in VEGF expression and vascular density between these groups were observed. These results suggest that long-term adaptation of tumours to chronic hypoxia in vivo, while not affecting vascularity, does influence the functional status of the microvessels in favour of a more homogeneous perfusion.     

Photodynamic therapy creates fluence rate-dependent gradients in the intratumoral spatial distribution of oxygen

In photodynamic therapy (PDT), treatment efficacy may be reduced by the presence of pre-existing tumor hypoxia or by oxygen depletion during the therapy. Tumor oxygenation during PDT has been measured with needle electrodes, but the intratumoral distribution of this oxygen is not known. In the present study, the spatial distribution of hypoxia during PDT was quantified using the hypoxia-labeling marker EF3. Mice bearing radiation-induced fibrosarcoma tumors were treated with Photofrin-mediated PDT to a total dose of 135 J/cm(2), delivered at a fluence rate of either 75 mW/cm(2) or 38 mW/cm(2). PDT-created hypoxia at each fluence rate was labeled by exposing tumors to EF3 (52 mg/kg) during the period of illumination. Cryosectioning, immunohistochemistry, and fluorescence microscopy were carried out to quantify EF3 binding as a function of distance to the nearest perfused blood vessels in sections cut from within the superficial (light-adjacent) 600 micro m or the deep (light-distant) 600 micro m of tumors (5-6 mm in diameter, approximately 3 mm in depth). In both superficial and deep sections, PDT at 75 mW/cm(2) resulted in the development of significant gradients in tumor hypoxia as a function of distance to a perfused blood vessel. Furthermore, significant hypoxia was detected even in vascular-adjacent tissue. These effects were associated with a significant decrease in the percentage of perfused vessels and a significant increase in the median distance of a cell to the nearest perfused blood vessel. In contrast, during PDT at 38 mW/cm(2), sections from deep tumor levels demonstrated only insignificant increases in the rise in hypoxia as a function of distance to a perfused vessel and in the level of hypoxia in vascular-adjacent tissue. No effects on tumor perfusion were detected during PDT at 38 mW/cm(2). Overall, these results demonstrate that spatially dependent depletion of oxygen can occur during PDT as a function of the fluence rate and that PDT can create significant hypoxia in even tissue adjacent to perfused blood vessels.     

Zonal image analysis of tumour vascular perfusion,hypoxia, and necrosis

A number of laboratories are utilising both hypoxia and perfusion markers to spatially quantify tumour oxygenation and vascular distributions, and scientists are increasingly turning to automated image analysis methods to quantify such interrelationships. In these studies, the presence of regions of necrosis in the immunohistochemical sections remains a potentially significant source of error. In the present work, frozen MCa-4 mammary tumour sections were used to obtain a series of corresponding image montages. Total vessels were identified using CD31 staining, perfused vessels by DiOC(7) staining, hypoxia by EF5/Cy3 uptake, and necrosis by haematoxylin and eosin staining. Our goal was to utilise image analysis techniques to spatially quantitate hypoxic marker binding as a function of distance from the nearest blood vessel. Several refinements to previous imaging methods are described: (1) hypoxia marker images are quantified in terms of their intensity levels, thus providing an analysis of the gradients in hypoxia with increasing distances from blood vessels, (2) zonal imaging masks are derived, which permit spatial sampling of images at precisely defined distances from blood vessels, as well as the omission of necrotic artifacts, (3) thresholding techniques are applied to omit holes in the tissue sections, and (4) distance mapping is utilised to define vascular spacing.     

Intravascular HBO(2) saturations, perfusion and hypoxia in spontaneous and transplanted tumor models

Clinical trials utilizing strategies to manipulate tumor oxygenation, blood flow and angiogenesis are under way, although limited quantitative information exists regarding basic tumor pathophysiology. The current study utilized murine KHT fibrosarcomas, spontaneous mammary carcinomas and first-generation spontaneous transplants to examine heterogeneity in vascular structure and function, to relate these changes to the distribution of tumor hypoxia and to determine whether fundamental relationships among the different pathophysiological parameters exist. Three methods were included: (i) immunohistochemical staining of anatomical and perfused blood vessels, (ii) cryospectrophotometric measurement of intravascular oxyhemoglobin saturations and (iii) fluorescent detection of the EF5 hypoxic marker. While a distinct pattern of decreasing oxygenation with increasing distance from the tumor surface was observed for KHT tumors, striking intertumor variability was found in both spontaneous and first-generation transplants, with a reduced dependence on tumor volume. EF5 hypoxic marker uptake was also much more heterogeneous among individual spontaneous and first-generation tumors compared to KHT. Although mammary carcinomas demonstrated fewer anatomical blood vessels than fibrosarcomas, the proportion of perfused vessels was substantially reduced in KHT tumors, especially at larger tumor volumes. Vascular morphology, tissue histological appearance and pathophysiological parameters differed substantially between KHT tumors and both spontaneous and first-generation tumors. Such differences in vascular structure and function are also likely to correlate with altered response to therapies targeted to the vascular system. Finally, spontaneous differentiation status, tumor morphology, vascular configuration and function were well preserved in first-generation transplanted tumors, suggesting a close relationship between vascular development and function in early-generation transplants and spontaneous tumor models.     

Sunitinib treatment does not improve blood supply but induces hypoxia in human melanoma xenografts

ABSTRACT: BACKGROUND: Antiangiogenic agents that disrupt the vascular endothelial growth factor pathway have been demonstrated to normalize tumor vasculature and improve tumor oxygenation in some studies and to induce hypoxia in others. The aim of this preclinical study was to investigate the effect of sunitinib treatment on the morphology and function of tumor vasculature and on tumor oxygenation. METHODS: A-07-GFP and R-18-GFP human melanoma xenografts grown in dorsal window chambers were used as preclinical tumor models. Morphologic parameters of tumor vascular networks were assessed from high-resolution transillumination images, and tumor blood supply time was assessed from first-pass imaging movies recorded after a bolus of 155 kDa tetramethylrhodamine isothiocyanate-labeled dextran had been administered intravenously. Tumor hypoxia was assessed from immunohistochemical preparations of the imaged tissue by use of pimonidazole as a hypoxia marker. RESULTS: Sunitinib treatment reduced vessel densities, increased vessel segment lengths, did not affect blood supply times, and increased hypoxic area fractions. CONCLUSION: Sunitinib treatment did not improve vascular function but induced hypoxia in A-07-GFP and R-18-GFP tumors.     

Selective cytokine inhibitory drugs with enhanced antiangiogenic activity control tumor growth through vascular inhibition

Selective cytokine inhibitory drugs (SelCIDs) are a novel class of phosphodiesterase 4 inhibitors discovered during a thalidomide analog discovery program. These analogs were evaluated for their ability to inhibit tumor angiogenesis, vascularity, and growth. Two analogs (CC-7034 and CC-9088) were identified that had enhanced antiangiogenic activity in Matrigel assays compared with parental thalidomide. These analogs also inhibited the growth of established K1735 and RENCA murine tumors. Tumors whose growth was suppressed by SelCID treatment exhibited decreased vessel density together with increased tumor cell hypoxia and death. The decrease in vascularity produced by SelCID treatment is attributed to a selective loss of vessels devoid of pericyte coverage, suggesting that these agents target immature tumor vessels. That tumor cell death was localized to relatively avascular or hypoxic areas, coupled with the fact that none of the analogs was cytotoxic in vitro against the tumor cells, demonstrates that these analogs are novel antivascular agents with potent antitumor activity.     

Changes in tumor hypoxia induced by mild temperature hyperthermia as assessed by dual-tracer immunohistochemistry

PURPOSE: To study the changes in hypoxia resulting from mild temperature hyperthermia (MTH) in a subcutaneous xenograft model using dual-tracer immunohistochemical techniques. MATERIALS AND METHODS: HT29 tumors were locally heated at 41 degrees C. Changes in tumor hypoxia were investigated by pimonidazole and EF5. Pimonidazole was given 1h preheating, EF5 at various times during or after treatment, 1h later the animals were sacrificed. Blood vessels were identified by CD31 staining, and perfusion by Hoechst 33342 injected 1min pre-sacrifice. RESULTS: The overall hypoxic fraction was significantly decreased by MTH during and immediately after heating. However, MTH induced both increases and decreases in tumor hypoxia in different parts of the tumor. Specifically, MTH decreased hypoxia in the regions with relatively well-perfused blood vessels, but increased hypoxia in regions that were poorly perfused. At 24-h post heating, newly formed hypoxic regions surrounded previously-hypoxic foci, which in turn surrounded pimonidazole-stained debris. Quantitative analysis did not evince changes in tumor oxygenation due to MTH at 24h post-treatment. CONCLUSION: In this xenograft model, the effect of MTH on tumor oxygenation was variable, both spatially and kinetically. Overall tumor oxygenation was improved during and after heating, but the effect was short-lived.     

Tumor oxygen dynamics: correlation of in vivo MRI with histological findings

Tumor oxygenation has long been recognized as a significant factor influencing cancer therapy. We recently established a novel magnetic resonance in vivo approach to measuring regional tumor oxygen tension, FREDOM (Fluorocarbon Relaxometry Using Echo Planar Imaging for Dynamic Oxygen Mapping), using hexafluorobenzene (HFB) as the reporter molecule. We have now investigated oxygen dynamics in the two Dunning prostate R3327 rat tumor sublines, AT1 and H. FREDOM revealed considerable intratumoral heterogeneity in the distribution of pO(2) values in both sublines. The anaplastic faster-growing AT1 tumors were more hypoxic compared with the size-matched, well-differentiated, and slower-growing H tumors. Respiratory challenge with oxygen produced significant increases in mean and median pO(2) in all the H tumors (P<.001), but no response in half of the larger AT1 tumors (>3 cm(3)). Immunohistochemical studies using the hypoxia marker, pimonidazole, and the vascular endothelial cell marker, CD31, confirmed that the H tumors had more extensive vasculature and less hypoxia than the AT1 tumors. These results further validate the utilization of FREDOM to monitor tumor oxygenation and concur with the hypothesis that the level of hypoxia is related to tumor growth rate and poor vascularity.     

Carbonic anhydrase 9 as an endogenous marker for hypoxic cells in cervical cancer.

The presence of radiation-resistant hypoxic cells in some solid tumors is known to predict for relapse after radiotherapy. Use of an endogenous marker of hypoxia would be a convenient alternative to current methods that measure tumor oxygenation, provided the marker could be shown to reliably identify viable, radiation-resistant, hypoxic cells. Carbonic anhydrase 9 (CA9) is a transmembrane protein overexpressed in a wide variety of tumor types and induced by hypoxia. Using a monoclonal antibody and cell sorting, CA9-positive cells in SiHa cervical carcinoma xenografts growing in immunodeficient mice were found to be clonogenic, resistant to killing by ionizing radiation, and preferentially able to bind the hypoxia marker pimonidazole. CA9 and pimonidazole immunostaining were compared in formalin-fixed sections from tumors of 18 patients undergoing treatment for cancer of the cervix. Excellent colocalization was observed, although the area of the tumor section that bound anti-CA9 antibodies represented double the number of cells that bound anti-pimonidazole antibodies. Occasional regions staining with pimonidazole but not CA9 could be indicative of transient changes in tumor perfusion. Results support the hypothesis that CA9 is a useful endogenous marker of tumor hypoxia.     

Thymidine analogues to assess microperfusion in human tumors

PURPOSE: To validate the use of the thymidine analogues as local perfusion markers in human tumors (no labeling indicates no perfusion) by comparison with the well-characterized perfusion marker Hoechst 33342. METHODS AND MATERIALS: Human tumor xenografts from gliomas and head-and-neck cancers were injected with iododeoxyuridine (IdUrd) or bromodeoxyuridine (BrdUrd) and the fluorescent dye Hoechst 33342. In frozen sections, each blood vessel was scored for the presence of IdUrd/BrdUrd labeling and Hoechst in surrounding cells. The percentage of analogue-negative vessels was compared with the fraction of Hoechst-negative vessels. Collocalization of the two markers was also scored. RESULTS: We found considerable intertumor variation in the fraction of perfused vessels, measured by analogue labeling, both in the human tumor xenografts and in a series of tumor biopsies from head-and-neck cancer patients. There was a significant correlation between the Hoechst-negative and IdUrd/BrdUrd-negative vessels in the xenografts (r = 85, p = 0.0004), despite some mismatches on a per-vessel basis. CONCLUSIONS: Thymidine analogues can be successfully used to rank tumors according to their fraction of perfused vessels. Whether this fraction correlates with the extent of acute hypoxia needs further confirmation.     

Vascular perfusion and hypoxic areas in RIF-1 tumours after photodynamic therapy.

The influence of photodynamic therapy (PDT) on vascular perfusion and the development of hypoxia was investigated in the murine RIF-1 tumour. Image analysis was used to quantify changes in perfusion and hypoxia at 5 min after interstitial Photofrin-mediated PDT. The fluorescent stain Hoechst 33342 was used as an in vivo marker of functional vascular perfusion and the antibody anti-collagen type IV as a marker of the tumour vasculature. The percentage of total tumour vasculature that was perfused decreased to less than 30% of control values after PDT. For the lower light doses this decrease was more pronounced in the centre of the tumour. The observed reduction in vascular perfusion showed a good linear correlation (r = 0.98) with previously published tumour perfusion data obtained with the 86Rb extraction technique. The image analysis technique provides extra information concerning the localisation of (non)-perfused vessels. To detect hypoxic tumour areas in vivo, an immunohistochemical method was used employing NITP [7-(4''-(2-nitroimidazol-1-yl)-butyl)-theophylline]. A large increase in hypoxic areas was found for PDT-treated tumours. More than half the total tumour area was hypoxic after PDT, compared with < 4% for control tumours. Our studies illustrate the potential of image analysis systems for monitoring the functional consequences of PDT-mediated vascular damage early after treatment. This provides direct confirmation that the perfusion changes lead to tissue hypoxia, which has implications for the combined treatment of PDT with bioreductive drugs.     

Acute hypoxia enhances spontaneous lymph node metastasis in an orthotopic murine model of human cervical carcinoma

An orthotopic mouse model of cervical carcinoma has been used to investigate the relationship between acute (cyclic) hypoxia and spontaneous lymph node metastasis in vivo. The human cervical carcinoma cell line ME-180 was stably transfected to express the fluorescent protein DsRed2, which allowed the in vivo optical monitoring of tumor growth and metastasis by fluorescent microscopy. The surgically implanted primary tumors metastasize initially to local lymph nodes and later to lung, a pattern consistent with the clinical course of the disease. The effect of acute hypoxia on the growth and spread of these tumors was examined by exposing tumor-bearing mice to treatment consisting of exposure to 12 cycles of 10 min 7% O(2) followed by 10 min air (total 4 h) daily during tumor growth. After 21 days, the tumors were excised, lymph node and lung metastases were quantified, and the hypoxic fraction and relative vascular area of the primary tumors were assessed by immunohistochemical staining for the hypoxic marker drug EF5 [2-(2-nitro-1H-imidazole-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] and the vascular marker CD31, respectively. In untreated mice, the primary tumor size was directly correlated with lymph node metastatic burden. The acute hypoxia treatment resulted in a significant decrease in the size of the primary tumors at the time of excision. However, the mice in the acute hypoxia group had an increased number of positive lymph nodes (2-4) as compared with control mice (1-3). Lung metastasis was not affected. The acute hypoxia treatment also decreased the relative vascular area in the primary tumors but did not affect the hypoxic fraction. These results suggest that fluctuating oxygenation in cervical carcinoma tumors may reduce tumor growth rate, but it may also enhance the ability of tumor cells to metastasize to local lymph nodes.     

Doppler ultrasound imaging detects changes in tumor perfusion during antivascular therapy associated with vascular anatomic alterations

Noninvasive monitoring of antiangiogenic therapy was performed by serial power Doppler ultrasound imaging of murine tumors treated with recombinant interleukin 12, the results of which were correlated with assessments of tumor vascularity by microscopy. Growth of established K1735 tumors, but not of IFN-gamma-unresponsive K1735.N23 variants, was suppressed by treatment. Serial Doppler imaging of K1735 tumor vascularity during treatment revealed a progressive change from a diffuse perfusion pattern to a more punctate distribution. Quantitative analysis of the images revealed that color-weighted fractional average, representing overall tumor perfusion, consistently decreased in these tumors, primarily because of a decrease in fractional tumor cross-sectional area carrying blood flow. In contrast, these parameters increased in nonresponsive tumors during treatment. Confocal microscopy of thick tumor sections revealed a reduction in the density and arborization of vessels labeled in vivo by fluorochrome-conjugated lectin with effective treatment. Immunohistological examination of thin tumor sections confirmed the preferential loss of small vessels with successful therapy. Similar changes in tumor vascular anatomy and perfusion were also observed during recombinant interleukin 12 treatment of two other responsive murine tumor types. These results indicate that power Doppler ultrasound is a sensitive, noninvasive method for reporting functional consequences of therapy-induced vascular anatomical changes that can be used to serially monitor tumor perfusion and efficacy of antivascular therapy in clinical trials.     

Characterization of the effects of antiangiogenic agents on tumor pathophysiology

A variety of strategies have been proposed to control tumor growth and metastasis by inhibiting tumor angiogenesis. To optimally combine such antiangiogenic approaches with conventional therapy, improved methods are needed to characterize the underlying pathophysiologic changes. The objective of the current work was to demonstrate the utility of a combination of recently developed immunohistochemical and image analysis techniques in quantitating changes in tumor vasculature and hypoxia. Murine MCa-35 mammary carcinomas were frozen after administration of two COX-2 inhibitors: meloxicam and celecoxib (Celebrex). Total blood vessels were visualized using anti-CD31 staining, perfused vessels by intravenous injection of DiOC7, and tumor hypoxia by EF5 uptake. Although both agents produced similar reductions in tumor volume compared with untreated tumors, varied effects on tumor vasculature and hypoxia were noted. Meloxicam reduced total vessel numbers significantly, whereas celecoxib had no effect. Both drugs substantially increased perfused vessel densities. Although mean hypoxic marker uptake was unchanged from matched controls, intratumor EF5 heterogeneities were significantly different between drugs. The results suggest that COX-2 inhibitors can have varying effects on tumor pathophysiology. Successful use of these drugs to enhance radiation response will likely require optimization of drug choice, dose schedule, and direct physiologic monitoring.     

Targeted anti-vascular endothelial growth factor receptor-2 therapy leads to short-term and long-term impairment of vascular function and increase in tumor hypoxia

Because antiangiogenic therapies inhibit the growth of new tumor-associated blood vessels, as well as prune newly formed vasculature, they would be expected to reduce the supply of oxygen and thus increase tumor hypoxia. However, it is not clear if antiangiogenic treatments lead only to consistent and sustained increases in hypoxia, or transient decreases in tumor hypoxia along with periods of increased hypoxia. We undertook a detailed analysis of an orthotopically transplanted human breast carcinoma (MDA-MB-231) over a 3-week treatment period using DC101, an anti-vascular endothelial growth factor receptor 2 antibody. We observed consistent reductions in microvascular density, blood flow (measured by high-frequency micro-ultrasound), and perfusion. These effects resulted in an increase in the hypoxic tumor fraction, measured with an exogenous marker, pimonidazole, concurrent with an elevation in hypoxia-inducible factor-1alpha expression, an endogenous marker. The increase in tumor hypoxia was evident within 5 days and remained so throughout the entire course of treatment. Vascular perfusion and flow were impaired at days 2, 5, 7, 8, 14, and 21 after the first injection, but not at 4 hours. A modest increase in the vessel maturation index was detected after the 3-week treatment period, but this was not accompanied by an improvement in vascular function. These results suggest that sustained hypoxia and impairment of vascular function can be two consistent consequences of antiangiogenic drug treatment. The implications of the results are discussed, particularly with respect to how they relate to different theories for the counterintuitive chemosensitizing effects of antiangiogenic drugs, even when hypoxia is increased.     

Effects of nicotinamide and carbogen in different murine colon carcinomas: immunohistochemical analysis of vascular architecture and microenvironmental parameters

PURPOSE: To investigate oxygenation, perfusion, and cell proliferation in two murine colon carcinoma lines with known differences in chemotherapy sensitivity and analyze the effect of nicotinamide and carbogen on these tumor characteristics. METHODS AND MATERIALS: Mice with s.c. transplanted C38 and C26a murine colon tumors were treated with nicotinamide and carbogen and compared with control tumors. Two markers of hypoxia, CCI-103F and pimonidazole, were injected before and after treatment with nicotinamide/carbogen, respectively, allowing each tumor to serve as its own control. Hoechst33342 was used as a perfusion marker and bromodeoxyuridine (BrdUrd) as a proliferation marker. Frozen tumors were cut for multistep immunostaining and computer-controlled microscope scanning for hypoxic fractions (HF), perfused fractions (PF), vascular density, and BrdUrd-labeling index (LI). RESULTS: Microscopic observation of C38 and C26a tumors showed extensive differences in vascular architecture, distribution patterns of hypoxia, and BrdUrd-labeling. Quantitative analysis of C38 and C26a tumors showed a decrease in HF in response to all treatment modalities. For C38 tumors, the average decrease in HF in response to carbogen containing treatments was larger than to nicotinamide alone. In C26a tumors, no difference in average decrease in HF was observed between the treatments. The PF of C38 and C26a did not change in response to treatment. The LI of C38 and C26a decreased upon all treatments, which was statistically significant in the combination treatment of C38. CONCLUSIONS: The mechanism that can simultaneously explain all the observed changes in response to treatment may be the conversion of metabolism from less respiration toward more glycolysis due to increased glucose levels (Crabtree effect), although other mechanisms of actions cannot be excluded.