Relative Vascular Area Research Articles

QS11. Proteasome Inhibitors Effectively Inhibit Venous and Lymphatic Malformations

Purpose: Venous and lymphatic malformations (VMs and LMs) carry postzygotic somatic mutations in malformation endothelial cells (VMECs, LMECs), leading to PI3K/AKT/mTOR hyperactivation.Off-label treatment with sirolimus, an mTOR inhibitor, and alpelisib, a PI3K inhibitor, has been effective, confirming the validity of a pharmacological approach in the treatment of VMs and LMs. However, treatment response is incomplete. We hypothesized that a high-throughput screen (HTPS) against VMECs and LMECs carrying PIK3CA mutations would identify novel candidate drugs to be used in the treatment of VMs and LMs. Methods: FDA-approved/in-clinical-trial drugs at 1uM concentration, and percent viability relative to vehicle-treated controls was determined at 48 hours. A dose response viability assay was performed to compare effective inhibitors to sirolimus and alpelisib. In a xenograft mouse model, VMECs (n=2) and LMECs (n=2) carrying PIK3CA variants were resuspended in Matrigel, which were subcutaneously implanted into the flanks of nude mice. Malformations were allowed to develop for either one week (VMs) or two weeks (LMs) before beginning randomized treatment with either oprozomib (a proteasome inhibitor) or a vehicle. Oprozomib was administered by mouth at 30mg/kg 2x per week for 4 weeks. Implants were harvested and analyzed histologically and by immunofluorescence, and relative vascular areas to total implant areas were measured. Statistical analysis was performed with one-way ANOVA with posthoc Tukey’s HSD as well as student’s t-test. Results: HTPS identified three proteasome inhibitors (PIs) that significantly suppressed VMEC and LMEC viability when compared to sirolimus and alpelisib: carfilzomib, delanzomib, and ixazomib (p<0.0001, ANOVA). In a dose response viability assay, all 6 PIs significantly inhibited VMEC and LMEC at clinically relevant doses (p<0.005, student’s t-test. Oprozomib was well-tolerated by mice in the xenograft model and effectively inhibited VM and LM development. When oprozomib-treated implants were compared to controls, LM channel sizes were significantly decreased (p<0.02), and relative vascular area of VM implants were also significantly decreased (p<0.007, student’s t-test). Conclusion: We identified a novel class of drugs, proteasome inhibitors, that significantly inhibited VMEC and LMEC viability in vitro and in vivo. These PIs had superior efficacy in VMEC and LMEC inhibition relative to sirolimus and alpelisib. These data demonstrate that proteasome inhibitors may be efficacious in VM and LM treatment. Additional studies are needed to further understand mechanisms of PI efficacy.

On the Regulatory Capabilities of Thyroid Mast Cells in Thiamazole Model of Hypothyroidism and Infrared Laser Exposure

The regulatory effect of mast cells on the state of thyroid gland in hypothyroidism and laser therapy remains unclear. Aim: to study the secretory processes of mast cells in relationship with the indicators of functional activity of thyroid gland. Materials and methods. Experimental groups: (55 rats) 1) intact rats, 2) hypothyroidism (thiamazole 25mg/kg) 3) hypothyroidism and 0.5W laser exposure, 4) hypothyroidism and 2.0W laser exposure. Histological samples of the thyroid gland were removed on the 1, 7, and 30 days. Histological sections were stained with toluidine blue. Morphometric data analysis included descriptive statistics and non-parametric tests (Mann Whitney, Spearman correlation coefficient). Results. The increase in the granular saturation of mast cells and the average histochemical coefficient was observed in the hypothyroidism group, the degranulation index increased by day 30. After 0.5 W laser exposure, there was a decrease in the granular content in mast cells and an increase in the degranulation index; the granular saturation increased by day 30. After 2.0 W laser exposure, the content of granules in mast cells decreased on day 1, and on days 7 and 30 it was higher than in the hypothyroidism group; the degranulation index decreased by day 30. The correlation was revealed between the indicators of granule accumulation in a mast cell, the index of mast cell degranulation, the thyroid epithelium height, and relative vascular area. Conclusions. The synthesis processes prevailed over secretion for mastocytes in thiamazole hypothyroidism. 0.5 W laser exposure was more effective for stimulation of the secretory processes in mast cells compared to 2.0 W exposure. The secretory activity of mast cells was associated with the functional activity of thyroid gland, which confirms their regulatory role in tissue repair after thiamazole induced hypothyroidism modeling.

Impact of light and sucrose supplementation on cellular differentiation, metabolic shift and modulation of gene expression in hairy roots of Daucus carota

Green hairy root cultures of Daucus carota were subjected to gradually lowered sucrose concentrations to understand the modulation of secondary metabolism upon carbon-starved conditions. An apparent negative correlation with the sucrose concentrations in the media was observed with chloroplast development, as confirmed by both histological studies and relevant biochemical assays. Expansion of relative vascular area and well-developed multi-layered endodermis were evidenced in green hairy roots with low sucrose concentrations as compared to normal hairy roots cultivated with 2% sucrose under dark condition. Thus, anatomical modulation was evident in photo-oxidatively stressed green hairy roots because of their maintenance under continuous illumination. This was demonstrated further by the expression analysis of glutathione peroxidase and NADPH reductase genes which showed upregulation in green hairy roots maintained in ½ MS media containing 0.5% sucrose as compared to normal hairy roots. The amount of phenolic marker compound p-hydroxybenzoic acid was reduced to half, while enhanced levels of emitted terpenoid volatiles were noticed in these carbon-starved green hairy roots. Analysis of primary metabolites in green hairy roots showed increased levels of amino acids and sugar alcohols accumulation. Further, analysis revealed the elevated accumulation of organic acids which have significant bearing towards redirection of primary metabolites leading to the formation of enhanced terpenoid volatiles in green hairy roots. Thus, our study revealed the impact of light and sucrose supplementation towards cellular differentiation and redirection of secondary metabolism in hairy roots of D. carota.

Correlation between arterial spin-labeling perfusion and histopathological vascular density of pediatric intracranial tumors.

Traditional MRI methods for estimation of blood flow in brain tumors require high-flow injection of contrast agents through large-bore intravenous access, which limits their pediatric utility. In contrast, arterial spin-labeling (ASL) can be used without contrast media. This study aimed to evaluate the relationship between tumor blood flow (TBF) measured by ASL and histopathological vascular density in pediatric brain tumors. Nineteen consecutive children were evaluated (10 boys, 9 girls; median age: 6 years; 8 high-grade and 11 low-grade tumors). ASL was performed with a pseudocontinuous labeling time of 1650ms and post-labeling delay of 1525ms. The maximal absolute (aTBF) and relative (rTBF) tumor blood flows were measured. To evaluate the relative vascular area (%Vessel), the total stained vascular area was divided by the whole tissue area. Spearman's rank-order correlation, the Mann-Whitney U test, and receiver operating characteristic analysis were used for statistical analysis. The absolute and relative TBF rates were 4.9-92.9mL/100g/min and 0.17-3.59mL/100g/min, respectively. The %Vessel was 0.6-30.2%. The %Vessel showed a significant positive correlation with TBF (aTBF: r = 0.87, P < 0.0001; rTBF: r = 0.89, P < 0.0001). The TBF rate of high-grade tumors was significantly higher than that of low-grade tumors (aTBF: P = 0.0050, rTBF: P = 0.0036). The rTBF had the best diagnostic performance (area under the curve: 0.89). ASL perfusion imaging without contrast material can be used for accurate evaluation of histopathological vascular density and may be helpful for tumor grading in children.

ΔNp63 isoform-mediated β-defensin family up-regulation is associated with (lymph)angiogenesis and poor prognosis in patients with squamous cell carcinoma.

Beside a role in normal development/differentiation, high p63 immunoreactivity is also frequently observed in squamous cell carcinoma (SCC). Due to the complexity of the gene, the role of each p63 isotype in tumorigenesis is still confusing. Constitutively produced or induced in inflammatory conditions, human beta-defensins (HβDs) are cationic peptides involved in host defenses against bacteria, viruses and fungi. Here, we investigated both the role of p63 proteins in the regulation of HβDs and the implication of these antimicrobial peptides in tumor (lymph)angiogenesis. Thus, in contrast to TAp63 isotypes, we observed that ΔNp63 proteins (α, β, γ) induce HβD1, 2 and 4 expression. Similar results were observed in cancer tissues and cell lines. We next demonstrated that ΔNp63-overexpressing SCC are associated with both a poor prognosis and a high tumor vascularisation and lymphangiogenesis. Moreover, we showed that HβDs exert a chemotactic activity for (lymphatic) endothelial cells in a CCR6-dependent manner. The ability of HβDs to enhance (lymph)angiogenesis in vivo was also evaluated. We observed that HβDs increase the vessel number and induce a significant increase in relative vascular area compared to negative control. Taken together, the results of this study suggest that ΔNp63-regulated HβD could promote tumor (lymph)angiogenesis in SCC microenvironment.

Lymphatic vascularization in primary breast cancer: HER2 overexpressing tumors contain more lymphatics than steroid receptor positive, triple-positive and triple negative breast carcinomas.

The aim of this study was to examine the relationship between the immunohistochemical subtypes of invasive breast cancer and lymphatic vascularization. One hundred and seventy nine cases of randomly selected invasive breast cancer patients, surgically treated between 2004 and 2007, were retrospectively studied. These were classified into steroid receptor positive (steroid receptor positive/ HER2 negative), triple positive (steroid receptor and HER2 positive), triple negative (steroid receptor and HER2 negative) and HER2 overexpressing (steroid receptor negative /HER2 positive) carcinomas. Appropriate immunostaining and in-situ hybridization techniques were applied and results were statistically analyzed. The median intra-tumor lymphatic vascular density and the median intra-tumor relative lymphatic vascular area were found to differ significantly among the studied groups of breast cancer (KW =49.8611; p < 0.0001 and KW =21.5122; p=0.0001 respectively). There was no significant difference in the incidence rate of axillary node involvement among the studied groups of breast cancer (χ < sup > 2 < /sup > =1.66; Df=3; p=0.6460). The present study indicates that HER2 overexpressing breast carcinomas have a consistent increase of intra-tumor lymphatic vessel counts as compared to all other subtypes. It is suggested that the newly formed vessels are probably not the only essential factor for lymphogenic spread of HER2 overexpressing breast carcinomas as they are not related to an increased incidence of lymph node metastases compared to the other studied subgroups.

Monitoring the effects of bevacizumab beyond progression in a murine colorectal cancer model: a functional imaging approach

Clinical studies have shown that bevacizumab beyond progression to first line therapy is beneficial for overall survival in advanced stage colorectal cancer. We studied the utility of several functional imaging modalities to assess the efficacy of bevacizumab beyond progression (BBP). All BALB/c mice with s.c. LS174T xenografts were treated with capecitabine, oxaliplatin and bevacizumab combination therapy. Tumor volume was assessed using caliper measurements. Increase of 1.5 times the initial volume on two subsequent measurements, was considered progression. In half of the mice bevacizumab treatment was continued (n = 13) after progressive disease was established, while the others received saline injections (n = 12). Within 3 days after progression, multi-modal imaging was performed using FDG-PET, diffusion weighted imaging, T2* and dynamic contrast enhanced MRI. Measurements were repeated 7 and 10 days after the first measurements. Afterwards, tumors were analyzed for expression of carbonic anhydrase IX, glucose transporter 1, 9 F1 to stain the vasculature and Ki67 to assess proliferation. In the BBP group tumor growth after progression was reduced compared to the control group (p < 0.01). FDG-PET showed a trend towards lower FDG uptake in the BBP group (p = 0.08). DWI, T2* and DCE-MRI parameters were not significantly different between both groups. The immunohistochemical analyses showed higher CAIX-positive fraction (p < 0.01) and lower Ki67 expression (p = 0.06) in the BBP group. The relative vascular area was significantly lower in the BBP group (p = 0.03). GLUT-1 expression and vascular density did not significantly differ between both groups. Bevacizumab after progression resulted in significant changes in the tumor proliferation and microenvironment compared to discontinuation of bevacizumab. FDG-PET may be sensitive to BBP-induced effects.

Reproducibility and biological basis of in vivo T2* magnetic resonance imaging of liver metastasis of colorectal cancer

In this study, the reproducibility of T2* MR imaging in colorectal liver metastases was assessed and T2* values were correlated with the expression of the hypoxia-related markers GLUT-1 and CA-IX as well as the relative vascular area, and the vessel density in resected tumors. The reproducibility of T2* was analyzed in 18 patients with in total 22 colorectal liver metastases using the Bland and Altman method for the 16th, 50th, and 84th percentile values. Immunohistochemical staining was performed on 17 resected tumors obtained from 16 patients. The median T2* of all liver metastases was 25.0 ± 5.6 ms vs. 23.0 ± 4.1 ms (median ± st.dev.) in normal liver. The coefficient of repeatability was 11.2 ms and the limits of agreement were -13.2 ms and 9.1 ms for median T2* values. On average, T2* showed fair reproducibility. No correlations between T2* values, hypoxia- and vascularity-related markers were observed.

Expression of EGFR Under Tumor Hypoxia: Identification of a Subpopulation of Tumor Cells Responsible for Aggressiveness and Treatment Resistance

Overexpression of epidermal growth factor receptor (EGFR) and tumor hypoxia have been shown to correlate with worse outcome in several types of cancer including head-and-neck squamous cell carcinoma. Little is known about the combination and possible interactions between the two phenomena. In this study, 45 cases of histologically confirmed squamous cell carcinomas of the head and neck were analyzed. All patients received intravenous infusions of the exogenous hypoxia marker pimonidazole prior to biopsy. Presence of EGFR, pimonidazole binding, and colocalization between EGFR and tumor hypoxia were examined using immunohistochemistry. Of all biopsies examined, respectively, 91% and 60% demonstrated EGFR- and pimonidazole-positive areas. A weak but significant association was found between the hypoxic fractions of pimonidazole (HFpimo) and EGFR fractions (F-EGFR) and between F-EGFR and relative vascular area. Various degrees of colocalization between hypoxia and EGFR were found, increasing with distance from the vasculature. A high fraction of EGFR was correlated with better disease-free and metastasis-free survival, whereas a high degree of colocalization correlated with poor outcome. Colocalization of hypoxia and EGFR was demonstrated in head-and-neck squamous cell carcinomas, predominantly at longer distances from vessels. A large amount of colocalization was associated with poor outcome, which points to a survival advantage of hypoxic cells that are also able to express EGFR. This subpopulation of tumor cells might be indicative of tumor aggressiveness and be partly responsible for treatment resistance.

Radiobiological hypoxia, histological parameters of tumour microenvironment and local tumour control after fractionated irradiation

Objective To investigate the relationships between radiobiological hypoxic fraction (rHF), pimonidazole hypoxic fraction (pHF) as well as other histological parameters of the tumour microenvironment, and local tumour control after fractionated irradiation in human squamous cell carcinomas (hSCCs). Material and methods Ten different hSCC cell lines were transplanted into nude mice and rHF was calculated from local tumour control rates after single dose irradiation under normal or clamped blood flow conditions. In parallel, tumours were irradiated with 30 fractions within 6 weeks. Radiation response was quantified as dose required to cure 50% of tumours (TCD 50). Unirradiated tumours were excised for histological evaluation including relative hypoxic area (pHF), relative vascular area (RVA), and fraction of perfused vessels (PF). Results A weak but significant positive correlation between rHF ( R 2 = 0.6, p = 0.014) and TCD 50 after fractionated irradiation was found. The pHF did not correlate with rHF but was significantly associated with the TCD 50 after single dose clamp ( R 2 = 0.8, p = 0.003) and showed a trend for an association with TCD 50 after fractionated irradiation ( R 2 = 0.4, p = 0.067). Relative vascular area and fraction of perfused vessels did not show an association with rHF or TCD 50 after fractionated irradiation. Conclusions Our data suggest that radiobiological hypoxia contributes to the response after fractionated irradiation but that also other radiobiological mechanisms are involved. In the present study, pimonidazole labelling does not reflect rHF and has a limited value to predict local tumour control after fractionated irradiation. The association between pHF and TCD 50 after single dose clamp warrants further investigation.

Assessment of Blood Hemodynamics by USPIO-Induced R 1 Changes in MRI of Murine Colon Carcinoma

The objective of this study is to assess whether ultrasmall superparamagnetic iron oxide (USPIO)-induced changes of the water proton longitudinal relaxation rate (R 1) provide a means to assess blood hemodynamics of tumors. Two types of murine colon tumors (C26a and C38) were investigated prior to and following administration of USPIO blood-pool contrast agent with fast R 1 measurements. In a subpopulation of mice, R 1 was measured following administration of hydralazine, a well-known blood hemodynamic modifier. USPIO-induced R 1 increase in C38 tumors (ΔR 1 = 0.072 ± 0.0081 s−1) was significantly larger than in C26a tumors (ΔR 1 = 0.032 ± 0.0018 s−1, N = 9, t test, P < 0.001). This was in agreement with the immunohistochemical data that showed higher values of relative vascular area (RVA) in C38 tumors than in C26a tumors (RVA = 0.059 ± 0.015 vs. 0.020 ± 0.011; P < 0.05). Following administration of hydralazine, a decrease in R 1 value was observed. This was consistent with the vasoconstriction induced by the steal effect mechanism. In conclusion, R 1 changes induced by USPIO are sensitive to tumor vascular morphology and to blood hemodynamics. Thus, R 1 measurements following USPIO administration can give novel insight into the effects of blood hemodynamic modifiers, non-invasively and with a high temporal resolution.

Tumor-Associated Lymphangiogenesis in the Development of Conjunctival Squamous Cell Carcinoma

To analyze whether tumor-associated lymphangiogenesis accompanies the development from premalignant conjunctival intraepithelial neoplasia (CIN) into invasive squamous cell carcinoma (SCC) of the conjunctiva and to study its association with prognosis and other tumor characteristics. Case-controlled, matched-pair cohort study. Twenty patients with invasive SCC were closely matched with 20 patients with high-grade CIN and 20 patients with low-grade CIN regarding tumor size, tumor location, tumor extension, and patients' age. Proliferating lymphatic vessels were identified using lymphatic vascular endothelial hyaluronan receptor-1 and podoplanin as specific lymphatic endothelial markers and Ki-67 as proliferation marker. Baseline tumor characteristics included tumor size, tumor-to-limbus distance, tumor-to-fornix distance, 2009 TNM classification, tumor cell type, mitotic rate, and Ki-67 proliferation index. Kaplan-Meier and Cox regression analyses of recurrence-free survival were performed. Lymphatic vascular density (LVD) and relative lymphatic vascular area (RLVA) of proliferating lymphatic vessels within the tumor mass (intratumoral) and within an area < or = 500 microm from the tumor border (peritumoral), and its association with tumor characteristics and recurrence-free survival. Intratumoral and peritumoral proliferating lymphatic vessels could be detected in all of the 60 conjunctival tumor samples. Invasive SCC revealed significantly higher values of intratumoral and peritumoral LVD and RLVA of proliferating lymphatics than high-grade or low-grade CIN (P < or = 0.001). Higher intratumoral lymphatic densities were significantly associated with larger tumor size (P=0.001), lower tumor-to-limbus distance (P=0.002), lower tumor-to-fornix distance (P=0.003), and higher TNM categories (P<0.001). Recurrence-free survival rates decreased significantly with higher intratumoral lymphatic densities (P<0.001). By multivariate Cox regression, large tumor size (hazard ratio 1.68, P=0.002) and high intratumoral lymphatic density (hazard ratio 1.10, P=0.046) were significant prognostic predictors of local recurrence. Development of conjunctival SCC from premalignant lesions is accompanied by the outgrowth of new conjunctival lymphatic vessels. This active tumor-associated lymphangiogenesis seems to be associated with an increased risk of local recurrence in patients with CIN and conjunctival invasive SCC. The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Hypoxia in larynx carcinomas assessed by pimonidazole binding and the value of CA-IX and vascularity as surrogate markers of hypoxia

Tumour hypoxia as driving force in tumour progression and treatment resistance has been well established. Assessment of oxygenation status of tumours may provide important prognostic information and improve selection of patients for treatment. In this study, a large homogenous group of 103 laryngeal carcinomas has been investigated in the presence of hypoxia by pimonidazole binding and the usefulness of Carbonic anhydrase IX (CA-IX) and vascular parameters as surrogate markers of hypoxia. These parameters are further related to clinical and biological characteristics. One hundred and three patients with T2–T4 larynx carcinoma were included. They were given the hypoxia marker pimonidazole intravenously (i.v.) 2 h prior to taking a biopsy. Expression of all the parameters was examined by immunohistochemistry, excluding large necrotic areas. Among tumours a large variation in pimonidazole positivity (hypoxic fraction based on pimonidazole, HFpimo) (range 0–19%) and CA-IX expression (hypoxic fraction based on CA-IX staining, HFCA-IX) (range 0–34%) was observed. In 67% of the tumours, hypoxia involved ⩾1% of the viable tumour area. HFpimo and HFCA-IX correlated significantly albeit weak ( p = 0.04). Both parameters showed weak inverse correlations with the relative vascular area (RVA) ( p = 0.01). HFpimo was further associated with histopathological grade, with poorly differentiated tumours being more hypoxic. The fraction of the tumour area positive for both pimonidazole and CA-IX correlated significantly with N stage. From these results, it was concluded that CA-IX and RVA have only limited value for measuring hypoxia and are not as robust as pimonidazole, probably due to the influence of other factors in the microenvironment. A combination of staining patterns of exogenous and endogenous markers might give important additive information about tumour biology and behaviour.

Core needle biopsies for determination of the microenvironment in individual tumours for longitudinal radiobiological studies

Purpose We aimed to establish a core needle biopsy technique to investigate the impact on outcome of irradiation of the microenvironment in individual experimental tumours. Methods Nude mice bearing FaDu, UT-SCC-5, UT-SCC-14, and UT-SCC-15 tumours ( n = 67) were injected with pimonidazole hypoxia and Hoechst 33342 perfusion markers. One core needle biopsy was taken from the central part of the tumour under anaesthesia and the rest of the tumour was excised after marking the position of the needle. Relative hypoxic area (pHF), relative vascular area (RVA), fraction of perfused vessels (PF), and necrotic fraction (NF) were compared in the biopsies and the adjacent whole tumour sections. In a TCD 50 (dose to cure 50% of tumours) assay, 223 UT-SCC-5 tumours were irradiated with 30 fractions over 6 weeks either with or without a core biopsy before the start of radiotherapy. Results The correlations between histological parameters measured in the biopsies and the adjacent tumour sections were dependent on the tumour line. All the four parameters showed weak although significant correlations only in UT-SCC-5. PF was the only parameter which showed a weak but significant correlation in all the four tumour lines. The needle biopsy procedure did not significantly impact on TCD 50 after fractionated irradiation in UT-SCC-5: 98 Gy [92; 106] versus 105 Gy [96; 117] ( p = 0.12). pHF, RVA, PF, and NF measured in the pre-treatment biopsy did not predict the outcome of fractionated irradiation within the UT-SCC-5 tumour line. Conclusion A single pre-treatment core needle biopsy may provide valid results for parameters of the tumour micromilieu, however the accuracy is limited by significant intratumoural heterogeneity in the parameters and sampling error. The needle biopsy procedure does not significantly affect local tumour control rates after fractioned irradiation and may therefore be integrated for longitudinal studies in radiobiological experiments. Pre-treatment histological parameters measured in the biopsy did not correlate with the outcome of fractionated irradiation within the UT-SCC-5 tumour line.

Effect of covalent antithrombin-heparin complex on developmental mechanisms in the lung

We have developed a potent antithrombin (AT)-heparin conjugate (ATH) that is retained in the lung to prevent pulmonary thrombosis associated with respiratory distress in premature newborns. During continuing maturation, pulmonary angiogenesis in premature infants would be a crucial process in lung development. A naturally occurring latent form of antithrombin (L-AT) has antiangiogenic effects on lung vascularization. However, impact of latent ATH (L-ATH) on developing lung vascularization is unknown. Thus, effects of L-AT and L-ATH on fetal murine lung development were compared. Lung buds from embryonic day 11.5 (E11.5) Tie2-LacZ mouse embryos were incubated in DMEM plus FBS supplemented with PBS, AT, L-AT, heparin, ATH, or L-ATH. Vasculature of cultured explants was quantified by X-galactosidase staining. RNA was analyzed with murine gene probes for angiopoietin (Ang)-1, Ang-2, fibroblast growth factor 2 (FGF2), platelet endothelial cell adhesion molecule (PECAM), and vascular endothelial growth factor (VEGF). FGF2-supplemented medium was used to test contribution to effects of L-AT and L-ATH on angiogenesis. Epithelial branching morphogenesis was inhibited by L-AT (P = 0.003) and heparin (P < 0.001). L-AT and heparin decreased relative vascular area compared with PBS, ATH, and L-ATH. Expressions of all genes studied were downregulated by L-AT. However, L-AT and L-ATH inhibited branching morphogenesis and vasculature with added FGF2. These findings indicate that covalent linkage of AT to heparin negates disruptive effects of these moieties on lung morphology, vascularization, and growth factor gene expression. ATH may have enhanced safety as an anticoagulant during vascular development.

Tumor Lymphangiogenesis in Transitional Cell Carcinoma of the Upper Urinary Tract: Association With Clinicopathological Features and Prognosis

Lymph node metastasis is an important prognostic factor in many types of cancer. Recently several specific markers for lymphatic endothelium were developed that facilitate the quantification of lymphangiogenesis in human cancer tissues. We investigated the clinical and prognostic significance of lymphangiogenesis in patients with transitional cell carcinoma of the upper urinary tract. We measured lymph vessel density and relative lymphatic vascular area in 125 specimens by quantitative immunohistochemical staining for D2-40 antibody (DakoCytomation, Glostrup, Denmark). These parameters were examined in the intratumor and peritumor areas, and measured using image analysis software. Peritumor lymph vessel density and peritumor lymphatic vascular area correlated with lymph node metastasis and tumor grade. In the intratumor area lymphatic vessels were detected in only 16.0% of specimens. However, the presence of intratumor lymphatic vessels was associated with lymph node metastasis (p = 0.002). Multivariate analysis identified high peritumor lymphatic vascular area and the presence of intratumor lymphatic vessels as significant and independent factors of metastasis-free survival after surgery (OR = 5.11, p = 0.020 and OR = 2.92, p = 0.025, respectively). Multivariate analysis also identified the presence of intratumor lymphatic vessels as the only independent predictive factor of cause specific survival (OR = 3.89, p = 0.049). Lymphangiogenesis may have important roles in tumor metastasis and survival in patients with transitional cell carcinoma of the upper urinary tract. Quantification of lymphatic vessels, especially peritumor lymphatic vascular area and intratumor lymphatic vessels, was useful for predicting metastasis-free survival. In addition, the presence of intratumor lymphatic vessels was an independent predictor of cause specific survival.

Carbogen Breathing Differentially Enhances Blood Plasma Volume and 5-Fluorouracil Uptake in Two Murine Colon Tumor Models with a Distinct Vascular Structure

For the systemic treatment of colorectal cancer, 5-fluorouracil (FU)-based chemotherapy is the standard. However, only a subset of patients responds to chemotherapy. Breathing of carbogen (95% O2 and 5% CO2) may increase the uptake of FU through changes in tumor physiology. This study aims to monitor in animal models in vivo the effects of carbogen breathing on tumor blood plasma volume, pH, and energy status, and on FU uptake and metabolism in two colon tumor models C38 and C26a, which differ in their vascular structure and hypoxic status. Phosphorus-31 magnetic resonance spectroscopy (MRS) was used to assess tumor pH and energy status, and fluorine-19 MRS was used to follow FU uptake and metabolism. Advanced magnetic resonance imaging methods using ultrasmall particles of iron oxide were performed to assess blood plasma volume. The results showed that carbogen breathing significantly decreased extracellular pH and increased tumor blood plasma volume and FU uptake in tumors. These effects were most significant in the C38 tumor line, which has the largest relative vascular area. In the C26a tumor line, carbogen breathing increased tumor growth delay by FU. In this study, carbogen breathing also enhanced systemic toxicity by FU.

Assessment of absolute blood volume in carcinoma by USPIO contrast-enhanced MRI

The characterization of tumor vasculature is essential in studying tumor physiology. The aim of this study was to develop a new method - based on water proton MR density measurements, in combination with ultrasmall superparamagnetic iron oxide (USPIO) administration - to measure absolute blood volume (BV) in murine colon carcinoma. MRI experiments were performed at 7 T. CPMG imaging was performed on subcutaneous murine colon carcinoma in six mice before and after administration of an USPIO blood-pool contrast agent. Density maps were obtained from the signal amplitude at TE=0 of the CPMG decay fit. Post-USPIO density maps were subtracted from pre-USPIO density maps to quantitatively yield absolute tumor BV maps. In a separate group of mice (n=6), the relative vascular area (RVA) of tumors was determined by immunohistochemistry. Ultrasmall superparamagnetic iron oxide administration resulted in a small decrease in the water proton MR density. The BV averaged over the six tumors was 4.6+/-1.6%. The value of the RVA measured by immunohistochemical staining was equal to 3.9+/-2.2%. After administration of an USPIO blood-pool agent (T(2) relaxivity > 100 mM(-1) s(-1)), the blood water protons become MRI invisible, and pixel-by-pixel BV map can be obtained by subtracting the calculated post-USPIO density map from the pre-USPIO density map. The value of absolute BV obtained with this novel MR approach is in good agreement with the value of the relative vascular measured by immunohistochemical staining.

Downregulation of TNF-alpha and VEGF expression by Sp1 decoy oligodeoxynucleotides in mouse melanoma tumor.

Melanoma tumor growth and progression are highly dependent on adequate blood supply through angiogenesis. Since several genes involved in angiogenesis revealed potential binding sites for the transcription factor Sp1, we have examined the effects of local inoculation of Sp1 decoy oligodeoxynucleotides (ODNs) on the growth of transplanted murine melanoma tumors and the expression of VEGF and TNF-alpha within these tumors. Treatment with Sp1 decoy ODNs, but not their mutated form, led to a significant increase (P=0.041) of the tumor necrotic area, as evaluated morphometrically. Tumor necrosis was associated with a significant decrease of microvascular density (P=0.012) and relative vascular area (P=0.026), as determined by counting CD34-positive vascular structures within the tumor microenvironment of Sp1 decoy ODNs and control ODN-treated tumors. RT-PCR experiments showed a strong decrease in the levels of VEGF188 and VEGF164 isoforms and a moderate decrease of TNF-alpha in Sp1 decoy-treated tumors. Taken together, our results indicate that Sp1 decoy ODNs may inhibit angiogenesis by affecting the gene expression of key players in angiogenesis such as TNF-alpha and VEGF. These findings indicate that Sp1 decoy ODNs may be a potential new therapeutic tool in antiangiogenic therapy.

Impact of the tumour bed effect on microenvironment, radiobiological hypoxia and the outcome of fractionated radiotherapy of human FaDu squamous-cell carcinoma growing in the nude mouse

Purpose : To investigate the impact of the tumour bed effect (TBE) on histological parameters of the micromilieu, radiobiological hypoxic fraction and local control after fractionated irradiation in FaDu squamous-cell carcinoma in the nude mouse. This tumour has previously shown a clear-cut TBE caused by increased necrotic cell loss at a constant cell production rate in the viable tumour compartment. Materials and methods : Human FaDu tumours were studied in the NMRI nude mouse. Tumours were transplanted either into unirradiated subcutaneous (s.c.) tissues (controls) or s.c. tissues pre-irradiated with 12.5 Gy (TBE group). In both groups we measured the volume doubling time (VDT), potential doubling time (T pot) , relative necrotic area, and in the viable tumour compartment the relative vascular area (9F1 mAb), relative hypoxic area (NITP or pimonidazole), relative perfused area (Hoechst 33342), and the perfused fraction of vasculature. The tumour control dose 50% (TCD 50), radiobiological hypoxic fraction (rHF) and dose-modifying factors (DMF) for the comparison of tumours in the TBE and control groups were determined from local tumour control data after treatment with single doses under ambient conditions or under clamp hypoxia, and after irradiation with 30 fractions under ambient conditions within 6 weeks using maximum-likelihood analysis. Results : A clear-cut TBE (VDT = 4.0 days (95%CI 2.9;4.4) for the control group versus 7.2 days (6.4;8.9) for the TBE group; p <0.0001) caused by increased necrosis (mean relative necrotic area of 12% (5;20)) versus 33% (10;41); p = 0.07) at a constant cell production rate (T pot = 2.2 days (1.4;2.3) versus 2.2 days (1.7;2.6); p = 0.30) was confirmed. Histological analysis of the micromilieu within the vital subarea revealed no systematic differences between the TBE and control groups. The rHF of 2% (0.1;27) for control tumours was lower than the 15% (95% CI 2;91) for the TBE group, but this difference was nonsignificant (p = 0.12). Compared with control tumours, the TCD 50 for irradiation under clamped hypoxia was in a statistical trend lower for tumours in the TBE group (DMF 1.11 (0.98;1.28), p = 0.09). After fractionated irradiation, tumours of the TBE group were significantly more radiosensitive (TCD 50 56.6 Gy (46;70) versus 78.7 Gy (63;100); p = 0.003). Conclusions : The results on FaDu tumours growing in pre-irradiated tissues indicate that increased necrosis caused by impairment of the vascular supply may increase the radiosensitivity of tumours treated by fractioned irradiation.