EGFR-inhibition enhances apoptosis in irradiated human head and neck xenograft tumors independent of effects on DNA repair.

Epidermal growth factor receptor (EGFR) inhibition using cetuximab improves the efficacy of radiotherapy in only a subgroup of head and neck squamous cell carcinoma (HNSCC) patients. Therefore, to improve patient selection a better understanding of tumor characteristics that affect treatment is necessary. Here, we investigated the effect of cetuximab on repair of radiation-induced DNA damage in a HNSCC xenograft model, which shows a synergistic effect to cetuximab and radiotherapy (SCCNij185) and a HNSCC model, which shows no additive effect of cetuximab to radiotherapy (SCCNij153). In both tumor models, clear increases were seen in the number of 53BP1 and Rad51 foci after irradiation. 53BP1 foci were present at comparable levels in hypoxic and normoxic tumor areas of the tumor xenografts, while the number of Rad51 foci was significantly higher in normoxic areas compared to hypoxic areas (P < 0.05). In both SCCNij185 and SCCNij153 xenografts an increased number of 53BP1 foci was observed in tumors treated with cetuximab and radiotherapy compared to radiotherapy alone. In SCCNij185 this increase was statistically significant in normoxic tumor areas (P = 0.04) and in SCCNij153 in both hypoxic and normoxic areas (P = 0.007 and P = 0.02, respectively). The number of Rad51 foci was not significantly different when cetuximab was added to radiotherapy compared to radiotherapy alone. Levels of pEGFR and pERK1/2 were decreased when cetuximab was added to radiotherapy in SCCNij185, but not in SCCNij153. Apoptosis was also only increased in SCCNij185 tumors at 4 days after cetuximab and radiotherapy treatment (P < 0.01). In conclusion, cetuximab inhibited DNA repair in both HNSCC models, but this effect was not predictive for the radiosensitizing effect of cetuximab in vivo. This lack of correlation may be related to differential effects of cetuximab and radiotherapy on ERK1/2 signaling and a decreased induction of apoptosis by cetuximab and radiotherapy in the resistant model.

Pimonidazole binding in C6 rat brain glioma: relation with lipid droplet detection.

In C6 rat brain glioma, we have investigated the relation between hypoxia and the presence of lipid droplets in the cytoplasm of viable cells adjacent to necrosis. For this purpose, rats were stereotaxically implanted with C6 cells. Experiments were carried out by the end of the tumour development. A multifluorescence staining protocol combined with digital image analysis was used to quantitatively study the spatial distribution of hypoxic cells (pimonidazole), blood perfusion (Hoechst 33342), total vascular bed (collagen type IV) and lipid droplets (Red Oil) in single frozen sections. All tumours (n=6) showed necrosis, pimonidazole binding and lipid droplets. Pimonidazole binding occurred at a mean distance of 114 microm from perfused vessels mainly around necrosis. Lipid droplets were principally located in the necrotic tissue. Some smaller droplets were also observed in part of the pimonidazole-binding cells surrounding necrosis. Hence, lipid droplets appeared only in hypoxic cells adjacent to necrosis, at an approximate distance of 181 microm from perfused vessels. In conclusion, our results show that severe hypoxic cells accumulated small lipid droplets. However, a 100% colocalisation of hypoxia and lipid droplets does not exist. Thus, lipid droplets cannot be considered as a surrogate marker of hypoxia, but rather of severe, prenecrotic hypoxia.

Quantitative analysis of varying profiles of hypoxia in relation to functional vessels in different human glioma xenograft lines.

Tissue oxygenation influences the radiation response of tumors. To further investigate the underlying mechanisms of tumor hypoxia, the spatial distribution of hypoxic cells in relation to the vasculature was studied. In a panel of three human glioma xenograft lines (E2, E102, E106) with different growth characteristics, tumor line-specific patterns of hypoxia (pimonidazole) and (functional) vasculature (Hoechst 33342) were observed. Two of the three glioma lines showed a more homogeneous distribution of perfused vessels (E102 and E106) than the third glioma line (E2). Although all tumors showed hypoxia, the distance at which the steepest part of the gradient of the hypoxia marker was found varied significantly among the different glioma lines. The faster-growing E102 tumors had the longest distance (>300 microm). These results indicate that tumor line-specific factors, rather than vascular geometry alone, may determine the oxygenation status of a tumor. As a consequence, vascular density cannot be used as a surrogate parameter for tumor hypoxia when comparing different tumors. Additional hypoxia and perfusion markers will further improve our understanding of changes in tumor physiology at the microregional level explaining the relationship between the low oxygen levels and the response of tumors to treatment.

Quantitation of the changes in vascularity during arthritis in the knee joint of a mouse with a digital image analysis system.

Many joint and bone diseases are caused by, or associated with vascular changes. Particularly in rheumatoid arthritis, vascular sprouting of synovial vessels plays a major role in the generation of joint pathology. To assess the effects of pharmaceuticals that are designed to inhibit neovascularization, we developed a quantitative procedure to measure vascular changes in cross-sections of the mouse knee joint during arthritic inflammation. Arthritis was induced in the knee joint of C57Black6 mice by a single subpatellar injection of methylated BSA after previous immunization. Total vascularity was visualized with a specific monoclonal rat anti-mouse antibody (9F1). Functional vessels were detected with the fluorescent perfusion marker Hoechst 33342. The localization of Hoechst and the vascular marker 9F1 were analyzed in separate images with an automated digital image processing system. By combining the two images, total vascularity and the perfusion status of the vessels during arthritis could be established. The digital image system measures synovial area (SA), number of all blood vessels (NBV) and the number of perfused blood vessels (NpBV). From these parameters the percentage of perfused vessels (perfusion fraction; PF), the vessel density (VD = NBV/SA) and the density of perfused vessels (VDp = NpBV/SA) can be calculated. The measurements showed that the area of synovial tissue had increased during arthritis. Moreover, both the number of blood vessels (NBV) and the number of perfused vessels (NpBV) in the synovial area had increased significantly on Days 4 and 7 after arthritis induction. This procedure enabled quantitation of total vascularity and of functional blood vessels in cross-sections of synovial tissue. It is expected to be a powerful tool, not only to analyze the effects of anti-angiogenic therapies in animal models of arthritis, but could also be applicable to study vascular and perfusion changes in vascular related diseases of the skeleton.

Changes in tumor hypoxia measured with a double hypoxic marker technique.

PURPOSE: Development of a double hypoxic cell marker assay, using the bioreductive nitroimidazole derivatives CCI-103F and pimonidazole, to study changes in tumor hypoxia after treatments that modify tumor oxygenation. METHODS AND MATERIALS: Both hypoxic markers were visualized by immunohistochemical techniques to detect changes in hypoxic fraction induced by carbogen breathing (95% O(2) and 5% CO(2)) or hydralazine injection. The protocol was tested in a human laryngeal squamous cell carcinoma xenograft line. Quantitative measurements were derived from consecutive tissue sections that were analyzed by a semiautomatic image analysis system. Qualitative analysis was obtained by double staining of the two hypoxic markers on the same tissue section. RESULTS: A significant correlation between the hypoxic fractions for the two markers, CCI-103F and pimonidazole, was found in air breathing animals. After carbogen breathing, the hypoxic fraction decreased significantly from 0.07 to 0.03, and after hydralazine treatment, the hypoxic fraction increased significantly. Reduction of hypoxia after carbogen breathing was most pronounced close to well-perfused tumor regions. CONCLUSIONS: With this method, employing two consecutively injected bioreductive markers, changes in tumor hypoxia can be studied. A significant reduction in hypoxia after carbogen breathing and a significant increase in hypoxia after hydralazine administration was demonstrated.

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.

Hypoxia in a human intracerebral glioma model.

OBJECT: The development of hypoxia in human gliomas is closely related to functional vasculature and the presence of hypoxia has important biological and therapeutic consequences. Assessment of hypoxia is necessary to understand its role in treatment response and to evaluate treatment strategies to improve tumor oxygenation. In this study, the authors report findings of their analysis of the degree of hypoxia in relation to other vascular parameters in a human intracerebral glioma xenograft. METHODS: In sections of tumor, hypoxic regions were identified immunohistochemically by using the hypoxic marker pimonidazole. The S-phase marker bromodeoxyuridine was used to detect cell proliferation, and the perfusion marker Hoechst 33342 was used to delineate perfused vessels. Vascular structures were stained with an endothelial marker. Hypoxic tumor regions were clearly present in this human intracerebral glioma model. Hypoxic areas were usually found in nonperfused regions, whereas tumor cell proliferation was especially marked in perfused tumor areas. Furthermore, by using in situ hybridization the authors identified infiltrating tumor cells in the normal brain. This feature is often observed in gliomas in patients. CONCLUSIONS: This model is a representative human glioma model that provides the researcher with the opportunity to analyze the relationship between the degree of hypoxia and vascular parameters, as well as to examine the effects of treatments aimed at modification of the oxygenation status of a tumor.

Vascular architecture and hypoxic profiles in human head and neck squamous cell carcinomas.

Tumour oxygenation and vasculature are determinants for radiation treatment outcome and prognosis in patients with squamous cell carcinomas of the head and neck. In this study we visualized and quantified these factors which may provide a predictive tool for new treatments. Twenty-one patients with stage III-IV squamous cell carcinomas of the head and neck were intravenously injected with pimonidazole, a bioreductive hypoxic marker. Tumour biopsies were taken 2 h later. Frozen tissue sections were stained for vessels and hypoxia by fluorescent immunohistochemistry. Twenty-two sections of biopsies of different head and neck sites were scanned and analysed with a computerized image analysis system. The hypoxic fractions varied from 0.02 to 0.29 and were independent from T- and N-classification, localization and differentiation grade. No significant correlation between hypoxic fraction and vascular density was observed. As a first attempt to categorize tumours based on their hypoxic profile, three different hypoxia patterns are described. The first category comprised tumours with large hypoxic, but viable, areas at distances even greater than 200 micrometer from the vessels. The second category showed a typical band-like distribution of hypoxia at an intermediate distance (50-200 micrometer) from the vessels with necrosis at greater distances. The third category demonstrated hypoxia already within 50 micrometer from the vessels, suggestive for acute hypoxia. This method of multiparameter analysis proved to be clinically feasible. The information on architectural patterns and the differences that exist between tumours can improve our understanding of the tumour micro-environment and may in the future be of assistance with the selection of (oxygenation modifying) treatment strategies.

Noninvasive assessment of the functional neovasculature in 9L-glioma growing in rat brain by dynamic 1H magnetic resonance imaging of gadolinium uptake.

Pathophysiologic parameters of the functional neovasculature and the blood-brain barrier of 9L-glioma in rat brain were measured noninvasively by dynamic 1H magnetic resonance imaging studies of gadolinium (Gd)-DTPA uptake. Changes of apparent [Gd-DTPA] uptake in time (CT[t]) were analyzed in a slice through the center of 10 9L-gliomas using fast T1 measurements. The distribution of the contrast agent was spatially correlated with the distribution of perfused microvessels as determined by immunohistochemical analysis. This method permits a distinction between perfused and nonperfused microvessels with a disrupted blood-brain barrier. In transverse slices of the whole tumor, a spatial correlation was observed between CT maps and the two-dimensional distribution of perfused microvessels. In the next step, Gd-DTPA uptake rates were spatially related to the perfused microvessel density (Np) or vascular surface area (Sp). In tumor voxels with perfused microvessels, a linear correlation was found between Gd-DTPA uptake rate constants (k values) and Np or Sp. No correlation was observed between k values and the total microvessel density. These are the first data that show a relation between Gd-DTPA uptake rates and parameters of the functional neovasculature in 9L-glioma growing in rat brain. Now that Gd-DTPA uptake studies can be related to parameters of the functional neovasculature, they may be used more efficiently as a prognostic tool before or during therapy.

Changes in blood perfusion and hypoxia after irradiation of a human squamous cell carcinoma xenograft tumor line.

The effect of irradiation depends on the oxygenation status of the tissue, while irradiation itself also changes the oxygenation and perfusion status of tissues. A better understanding of the changes in tumor oxygenation and perfusion over time after irradiation will allow a better planning of fractionated radiotherapy in combination with modifiers of blood flow and oxygenation. Vascular architecture (endothelial marker), perfusion (Hoechst 33342) and oxygenation (pimonidazole) were studied in a human laryngeal squamous cell carcinoma tumor line grown as xenografts in nude mice. The effect of a single dose of 10 Gy X rays on these parameters was evaluated from 2 h to 11 days after irradiation. Shortly after irradiation, there was an 8% increase in perfused blood vessels (from 57% to 65%) followed by a significant decrease, with a minimum value of 42% at 26 h after irradiation, and a subsequent increase to control levels at 7 to 11 days after irradiation. The hypoxic fraction showed a decrease at 7 h after treatment from 13% to 5% with an increase to 19% at 11 days after irradiation. These experiments show that irradiation causes rapid changes in oxygenation and perfusion which may have consequences for the optimal timing of radiotherapy schedules employing multiple fractions per day and the introduction of oxygenation- and perfusion-modifying drugs.

Global HDO uptake in human glioma xenografts is related to the perfused capillary distribution.

The aim of this study is to evaluate the existence of a possible relationship between global deuterium-labeled water (HDO) uptake rates and the diffusion geometry of human glioma xenografts in nude mice. HDO diffusion times in the whole extravascular tumor volume were estimated by combining quantitative (1)H-MR diffusion imaging and morphometric analysis of intercapillary distances in two tumor lines with a different perfused vascular architecture. HDO uptake was measured independently using (2)H-magnetic resonance spectroscopy. Time constants of HDO-uptake curves (tau) were compared to estimations of maximum HDO diffusion times (t(difmax)). Tumors with a homogeneously perfused capillary distribution showed a mono-exponential HDO uptake. The t(difmax) was comparable to tau values of HDO uptake curves: t(difmax) varied between 74 and 368 sec and the range of tau values was 115-370 sec. Heterogeneously perfused tumors had a bi-exponential HDO uptake with t(difmax) in between the tau values of the fast and slow uptake phase. These findings indicate that the global HDO uptake is related to the perfused capillary distribution in human glioma xenografts. That HDO uptake rates indeed can depend on the perfused capillary distribution was substantiated in experiments with two-dimensional (2D) models. In these models with a diffusion-limited HDO uptake, HDO uptake curves could be approximated by curves derived from 2D HDO diffusion simulations. Magn Reson Med 42:479-489, 1999.

Vascular architecture and microenvironmental parameters in human squamous cell carcinoma xenografts: effects of carbogen and nicotinamide.

BACKGROUND AND PURPOSE: A better understanding of the vascular architecture and the microenvironmental parameters (VAMP) will allow the identification of tumours that can be more effectively treated by intensified fractionated radiotherapy or modifiers of blood flow and oxygenation or combinations of these approaches. MATERIALS AND METHODS: Proliferation (BrdUrd), vascular architecture (endothelial marker), perfusion (Hoechst 33342) and oxygenation (NITP) were studied in two human laryngeal squamous cell carcinoma tumour lines grown as xenografts in nude mice. The effects of carbogen and nicotinamide on these parameters were evaluated. RESULTS: Carbogen treatment resulted in a decrease of the number of perfused blood vessels from 66% to 55% in one of the two tumour lines. In this tumour line nicotinamide prevented this reduction of tumour blood flow by carbogen. In both tumour lines the labelling index (LI) decreased after treatment with carbogen for 1 h, from 11-13% to 5-7%. Both tumour lines showed a drastic reduction of hypoxia by carbogen alone or by carbogen plus nicotinamide. CONCLUSIONS: In both laryngeal squamous cell carcinoma xenograft tumour lines carbogen was very effective in reducing diffusion limited hypoxia. Only in one of the two tested tumour lines carbogen also caused a reduction of tumour blood perfusion, which could be compensated for by nicotinamide. In addition, carbogen reduced tumour cell proliferation. The fact that differences in response to nicotinamide and carbogen were observed and that they can be studied in vivo provides a basis for further development of a 'predictive profile' which will guide the clinician to select the optimal treatment for individual patients or groups of patients.

Characterization and validation of noninvasive oxygen tension measurements in human glioma xenografts by 19F-MR relaxometry.

PURPOSE: The aim of this study was to characterize and to validate noninvasive 19F-magnetic resonance relaxometry for the measurement of oxygen tensions in human glioma xenografts in nude mice. The following three questions were addressed: 1. When perfluorocarbon compounds (PFCs) are administrated intravenously, which tumor regions are assessed by 19F-MR relaxometry? 2. Are oxygen tension as detected by 19F-MR relaxometry (pO2/relaxo) comparable to Eppendorf O2-electrode measurements (pO2/electrode)? 3. Can 19F-MR relaxometry be used to detect oxygen tension changes in tumor tissue during carbogen breathing? METHODS AND MATERIALS: Slice-selective 19F-MR relaxometry was carried out with perfluoro-15-crown-5-ether as oxygen sensor. The PFC was injected i.v. 3 days before the 19F-MR experiments. Two datasets were acquired before and two after the start of carbogen breathing. The distribution of PFCs and necrotic areas were analyzed in 19F-Spin Echo (SE) density MR images and T2-weighted 1H-SE MR images, respectively. One day after the MR investigations, oxygen tensions were measured by oxygen electrodes in the same slice along two perpendicular tracks. These measurements were followed by (immuno)histochemical analysis of the 2D distribution of perfused microvessels, hypoxic cells, necrotic areas, and macrophages. RESULTS: The PFCs mainly became sequestered in perfused regions at the tumor periphery; thus, 19F-MR relaxometry probed mean oxygen tensions in these regions throughout the selected MR slice. In perfused regions of the tumor, mean PO2/relaxo values were comparable to mean PO2/electrode values, and varied from 0.03 to 9 mmHg. Median pO2/electrode values of both tracks were lower than mean pO2/relaxo values, because low pO2 electrode values that originate from hypoxic and necrotic areas were also included in calculations of median pO2/electrode values. After 8-min carbogen breathing, the average PO2/relaxo increase was 3.3 +/- 0.8 (SEM) mmHg and 2.1 +/- 0.6 (SEM) after 14 min breathing. CONCLUSIONS: We have demonstrated that PFCs mainly became sequestered in perfused regions of the tumor. Here, mean PO2/relaxo values were comparable to mean PO2electrode values. In these areas, carbogen breathing was found to increase the PO2/relaxo values significantly.

A quantitative analysis of vascularization and perfusion of human glioma xenografts at different implantation sites.

The effect of tissue site of implantation of four different human gliomas on tumor vascularity and perfusion was examined. Vascular parameters of gliomas implanted subcutaneously in the nude mouse and intracerebrally in the nude rat were analyzed. Tumor vessels were stained with an antibody to collagen type IV and perfusion was investigated with the perfusion marker Hoechst 33342. Characteristic vascular patterns were observed in both intracerebral and subcutaneous xenografts belonging to the same tumor line. Major differences in vascular architecture and in the degree of vascularization were noted in comparisons of the two implantation sites for the same tumor line. Tumor perfusion was highly variable for both locations of tumor growth. Distinct differences between the implantation sites of similar tumor lines in vascular perfusion, intervascular distance, and vascular density were present. Incomplete perfusion of vascular structures, as seen in this study, may result in reduced delivery of oxygen to tumor areas. Therefore, measurements of vascular density and intervascular distance alone, without knowledge of the perfusion status, may not be sufficient to estimate the degree of tumor oxygenation. Furthermore, differences in vascular parameters may have important consequences for treatment modalities such as radiotherapy and chemotherapy. Thus, the findings in our study suggest that care has to be taken in extrapolating therapy results obtained with subcutaneous glioma tumor models to the original growth location of gliomas, the brain, due to major differences in vasculature.

Suramin treatment of human glioma xenografts; effects on tumor vasculature and oxygenation status.

In this study the effect of suramin on tumor growth, vascularity and oxygenation of a human glioma xenografted in the nude mouse was examined. Vascular parameters and oxygenation status of the xenografts were determined immunohistochemically in frozen sections of the tumors, using the hypoxia marker pimonidazole-hydrochloride to detect hypoxic areas. Tumor vessels in these sections were stained by an endothelial cell marker and perfusion of vessels was visualized by administration of the perfusion marker Hoechst 333342 before harvesting the tumors. The vascular parameters were quantified with an image analysis system. The results show that tumor growth was reduced considerably after suramin treatment. This growth suppression was accompanied by marked changes in vascular architecture. Although the total vascular area and perfused fraction of tumor vessels remained unchanged after suramin treatment, vascular density increased, indicating that more but smaller vessel structures had developed during therapy. These vessel structures were also more homogeneously spread over the tumor area. Control tumors showed extensive areas of hypoxia while in treated tumors hypoxic areas had mostly disappeared. This effect was probably due to the higher density of homogeneously distributed perfused vessel structures in the treated tumors, contributing to an increased oxygenation of the tumor. These observations suggest that suramin therapy can result in marked changes not only in tumor vascularity but also in tumor oxygenation status which may have important consequences for sensitivity of these tumors to other therapies such as radiation treatment.

Delayed vascular changes after antiangiogenic therapy with antivascular endothelial growth factor antibodies in human glioma xenografts in nude mice.

OBJECTIVE: The purpose of this study was to examine the delayed effects of antivascular endothelial growth factor treatment on tumor growth and vascularity in a subcutaneous mouse tumor model of human glioblastoma. METHODS: Antivascular endothelial growth factor antibody treatment was administered for a period of 6 weeks, to suppress tumor growth. To detect late vascular effects, tumor vascular parameters for treated tumors and control tumors were analyzed 4 weeks thereafter. By that time, tumors had grown to adequate sizes (diameter, 8-10 mm) for comparison with untreated control tumors. Vascular parameters were quantified by using an image-analysis system. RESULTS: Vascular density was significantly lower in antivascular endothelial growth factor antibody-treated tumors, compared with control tumors of similar size. The vascular architecture of treated tumors was also distinctly different, compared with control tumors, showing larger but sparser vessel structures. CONCLUSION: These findings suggest that antiangiogenic therapy may have a prolonged effect on the vascular architecture of certain tumors, resulting in enduring changes in the tumor vessels. Because tumor vasculature plays an important role in the sensitivity to various treatment modalities, these changes are likely to influence the responses of these tumors to further therapy.

The effect of the anti-angiogenic agent TNP-470 on tumor growth and vascularity in low passaged xenografts of human gliomas in nude mice.

The effect of the anti-angiogenic agent TNP-470 on tumor growth, vascular area, vascular density and tumor perfusion of two different subcutaneously implanted human glioma xenografts (E98 and E106) in nude mice was evaluated. Vascular parameters were investigated with an image analysis system. For both tumor lines a small but significant tumor growth suppression was observed. However, no differences in vascular parameters between TNP-470 treated tumors and controls could be found after 6 weeks of treatment. It is concluded that although TNP-470 is a promising anti-angiogenic agent in many tumor types, at least 2 glioma lines seem to be partly resistant to its anti-angiogenic effects. Further evaluation of the effects of combination of TNP-470 and cytostatic agents or radiotherapy in human glioma xenografts are required to determine the place of anti-angiogenic therapy in general and treatment with the anti-angiogenic agent TNP-470 more specifically in the treatment of human gliomas.

Multiparameter analysis of vasculature, perfusion and proliferation in human tumour xenografts.

A method is presented in this report for concurrent analysis of vascular architecture, blood perfusion and proliferation characteristics in whole-tumour cross-sections of human larynx carcinoma and glioblastoma xenografts. Tumours were implanted subcutaneously in nude mice. After i.v. injection with Hoechst 33342 and bromodeoxyuridine (BrdUrd) as perfusion and proliferation markers, animals were killed. An antiendothelial antibody (9F1) was used to delineate vascular structures. Cross-sections were analysed by a multistep immune staining and a computer-controlled microscope scanning method. Each tumour section was stained and scanned four times (Hoechst, 9F1, BrdUrd and Fast Blue for all nuclei). When these images were combined, vasculature, perfusion and proliferation parameters were analysed. The labelling index (LI) was defined as the ratio of the BrdUrd-labelled area to the total nuclear area. The LI based on manual counting and the LI calculated by flow cytometry (FCM) were in good agreement with the LI based on surface analysis. LI decreased at increasing distance from its nearest vessel. In the vicinity of perfused vessels, the LI was 30-70% higher than near non-perfused vessels. This method shows that both vasculature/perfusion and proliferation characteristics can be measured in the same whole-tumour section in a semiautomatic way. This could be applied in clinical practice to identify combined human tumour characteristics that predict for a favourable response to treatment modifications.

[Vascularization and perfusion of tumors as target in cancer therapy]. / Vascularisatie en doorbloeding van tumoren als doelwit voor kankertherapie.

Many tumours depend on the formation of an own vascular system to support progressive tumour growth. This is accomplished through induction of vessel growth from pre-existing vessels, a process called neo-angiogenesis. Therefore, inhibiting neo-angiogenesis and modulating tumour perfusion constitute attractive possibilities for tumour therapy, combined, of course, with treatment aimed at the tumour cells themselves. By now many angiogenesis inhibitors have been developed, but their use is mostly still experimental. They inhibit endothelial proliferation and migration (fumagillin derivates, angiostatin, suramin) or prevent proteolytic degeneration of the extracellular matrix by products of the tumour (metalloproteinase inhibitors). Improving tumour oxygenation and perfusion by carbogen inhalation and nicotinamide or vasoactive agents (flunarizine, verapamil, nicotinamide) enhances the effects of radiotherapy and improves delivery of chemotherapeutic agents to the tumour. Research is currently in progress into the efficacy of accelerated radiotherapy in combination with carbogen inhalation and administration of nicotinamide in tumours of the head and neck, bladder, bronchi and brain.

Quantitation of angiogenesis in healing anastomoses of the rat colon.

An accurate and reliable quantitation of angiogenesis is an essential requirement for a detailed study of new blood vessel growth during healing of colon anastomoses. In the present study we applied a computer-based digital image processing system for quantitative analysis of the anastomotic vascularization and perfusion. Rats underwent colonic resection (1 cm) followed by construction of an end-to-end anastomosis. The animals were killed 3 or 7 days after operation. The vascularization and perfusion were analysed in cyrostat sections using an anti-collagen type IV antibody and the fluorescent marker bisbenzimide H 33342, respectively. If compared with 3-day-old anastomoses, a significant increase in the median vascular and perfusion areas was found 7 days after operation. Within the same anastomosis, the vessel density and perfusion were lower in the central region than in the peripheral region of the wound. The present computerized digital image analysis system is a reliable technique that is suited for quantitative measurements of microvascular changes occurring in colon anastomoses during the first postoperative week.