Biological Principles of Stereotactic Body Radiation Therapy (SBRT) and Stereotactic Radiation Surgery (SRS): Indirect Cell Death.

PURPOSE: To review the radiobiological mechanisms of stereotactic body radiation therapy stereotactic body radiation therapy (SBRT) and stereotactic radiation surgery (SRS). METHODS AND MATERIALS: We reviewed previous reports and recent observations on the effects of high-dose irradiation on tumor cell survival, tumor vasculature, and antitumor immunity. We then assessed the potential implications of these biological changes associated with SBRT and SRS. RESULTS: Irradiation with doses higher than approximately 10 Gy/fraction causes significant vascular injury in tumors, leading to secondary tumor cell death. Irradiation of tumors with high doses has also been reported to increase the antitumor immunity, and various approaches are being investigated to further elevate antitumor immunity. The mechanism of normal tissue damage by high-dose irradiation needs to be further investigated. CONCLUSIONS: In addition to directly killing tumor cells, high-dose irradiation used in SBRT and SRS induces indirect tumor cell death via vascular damage and antitumor immunity. Further studies are warranted to better understand the biological mechanisms underlying the high efficacy of clinical SBRT and SRS and to further improve the efficacy of SBRT and SRS.

Characteristic Blood-Perfusion Reduction of Walker 256 Tumor Induced by Diagnostic Ultrasound and Microbubbles.

Tumor angiogenesis is characterized by a defective, leaky and fragile microvascular construction, and microbubble-enhanced ultrasound (MEUS) with high-pressure amplitude is capable of disrupting tumor microvasculature and arresting blood perfusion. In this study, we tried to investigate whether the blood perfusion of a malignant tumor can be characteristically interrupted by combining microbubbles and diagnostic ultrasound (US). Twenty-nine Sprague-Dawley (SD) rats with subcutaneous Walker 256 tumors and seven healthy SD rats were included. Fifteen tumors were treated by MEUS, which combined constant microbubble injection and 20 episodes of irradiation by diagnostic US (i.e., acoustic radiation force impulse [ARFI] imaging). The other 14 tumors were treated by ARFI or sham US only. Seven skeleton muscles from healthy SD rats were also treated with MEUS, serving as the control. Contrast-enhanced ultrasound (CEUS) was performed before and after all treatments. The blood perfusion of the tumor MEUS group showed a significant drop immediately after treatment, followed by a quick, incomplete perfusion recovery within 10-20 min. The visual perfusion scoring result was consistent with the quantitative analysis by CEUS peak intensity. However, there were no significant perfusion changes in the tumor control groups or the muscle control group. Histologic examination found severe microvascular disruption and hemorrhage in the MEUS-treated tumors but not in the control groups. Therefore, the treatment combining diagnostic US and microbubbles can specifically decrease or interrupt the blood perfusion of Walker 256 tumors, which could be a potential new imaging method for diagnosing malignant tumors.

Red propolis and L-lysine on angiogenesis and tumor growth in a new model of hamster cheek pouch inoculated with Walker 256 tumor cells.

OBJECTIVE: To evaluate the effect of red propolis and L-lysine on angiogenesis and tumor growth in a new model of hamster cheek pouch inoculated with Walker 256 tumor cells. METHODS: The study consisted of two experiments with four groups each (total: 57 hamsters). In the experiment 1, the animals were inoculated with Walker tumor cells, followed by administration of test substances (red propolis 200mg/5mL/kg or L-lysine 150mg/kg) or control substances (gum arabic 5mL/kg or water 5mL/kg) for 10 days. The animals in the experiment 2 received red propolis, L-lysine, gum arabic or water at the same doses, for 33 days prior to inoculation of Walker tumor cells, followed by 10 days of treatment with the same substances. Based on single-plane images, angiogenesis was quantified (mean vascular area), in percentage, and tumor area (mm2) and perimeter (mm). RESULTS: In the experiment 1, compared to animals receiving water, the mean vascular area expressed in percentage was significantly smaller in animal treated with propolis (p<0.05) and L-lysine (p<0.001). CONCLUSION: Both red propolis and L-lysine inhibited tumor angiogenesis in the new hamster cheek pouch model when administered after tumor inoculation.

Red propolis and L-lysine on angiogenesis and tumor growth in a new model of hamster cheek pouch inoculated with Walker 256 tumor cells / Própolis vermelha e L-lisina na angiogênese e no crescimento tumoral em novo modelo de bolsa jugal de hamster inoculada com células de tumor de Walker 256

ABSTRACT Objective: To evaluate the effect of red propolis and L-lysine on angiogenesis and tumor growth in a new model of hamster cheek pouch inoculated with Walker 256 tumor cells. Methods: The study consisted of two experiments with four groups each (total: 57 hamsters). In the experiment 1, the animals were inoculated with Walker tumor cells, followed by administration of test substances (red propolis 200mg/5mL/kg or L-lysine 150mg/kg) or control substances (gum arabic 5mL/kg or water 5mL/kg) for 10 days. The animals in the experiment 2 received red propolis, L-lysine, gum arabic or water at the same doses, for 33 days prior to inoculation of Walker tumor cells, followed by 10 days of treatment with the same substances. Based on single-plane images, angiogenesis was quantified (mean vascular area), in percentage, and tumor area (mm2) and perimeter (mm). Results: In the experiment 1, compared to animals receiving water, the mean vascular area expressed in percentage was significantly smaller in animal treated with propolis (p<0.05) and L-lysine (p<0.001). Conclusion: Both red propolis and L-lysine inhibited tumor angiogenesis in the new hamster cheek pouch model when administered after tumor inoculation.

RESUMO Objetivo: Avaliar o efeito da própolis vermelha e da L-lisina na angiogênese e no crescimento tumoral em novo modelo de bolsa jugal de hamster inoculada com células de tumor de Walker 256. Métodos: O estudo consistiu em dois experimentos com quatro grupos cada (total: 57 hamsters). No experimento 1, os animais foram inoculados com células de tumor de Walker, tendo em seguida administradas as substâncias teste (própolis vermelha 200mg/5mL/kg ou L-lisina 150mg/kg) ou controle (goma arábica 5mL/kg ou água 5mL/kg) por 10 dias. Os animais do experimento 2 receberam própolis vermelha, L-lisina, goma arábica ou água nas mesmas doses, por 33 dias antes do inóculo das células de tumor de Walker, seguido por 10 dias de tratamento com as mesmas substâncias. Baseado em imagens em plano único, foram quantificados a angiogênese (área vascular média), em termos percentuais, e a área (mm2) e o perímetro (mm) do tumor. Resultados: Comparada aos animais que receberam água, a área vascular média, expressa em percentagem, foi significativamente menor nos animais tratados com própolis (p<0,05) e com L-lisina (p<0,001). Conclusão: Tanto a própolis vermelha quanto a L-lisina inibiram a angiogênese no novo modelo de bolsa jugal de hamsters, quando administradas após a inoculação do tumor.

Walker 256/B malignant breast cancer cells improve femur angioarchitecture and disrupt hematological parameters in a rat model of tumor osteolysis.

This study was designed to assess femur angioarchitecture and hematological effects of Walker 256/B cells in a rat model of tumor osteolysis. Tumor osteolysis was induced by in situ inoculation of Walker 256/B malignant cells. Six other rats were sham operated and served as control. Twenty days later, rats were euthanized, and femurs were collected than radiographed. Angioarchitecture [mean lumen diameter (MLD), wall thickness (WTh), Vessel number, volume, and separation (VNb, VV, and VSp respectively)] was studied by histomorphometry at 2 different positions (P1: diaphysis, and P2: metaphysis) of the operated femora. Some hematological parameters were also assessed. Walker 256/B induced marked tumor osteolysis, with cortical perforation and trabecular destruction, associated increase in bone vascularization (increases of VNb and VV and decrease of VSp). Angioarchitecture of W256/B rats was disorganized and showed large MLD and lower WTh. These effects were more prominent in P2. When compared to Sham group, significantly decreases at levels of red blood cell (RBC), hemoglobin (Hb), hematocrit (Ht), and white blood cell (WBC) were observed in W256/B rats. These results suggest that Walker 256/B cells induced tumor osteolysis, improve hypervasculature especially near the tumoral foci (P2) associated hematological disruption. Besides, tumor vessels showed abnormal (enlarged and thinner) and disorganized morphology.

Asparagus polysaccharide and gum with hepatic artery embolization induces tumor growth and inhibits angiogenesis in an orthotopic hepatocellular carcinoma model.

Liver cancer is one of leading digestive malignancies with high morbidity and mortality. There is an urgent need for the development of novel therapies for this deadly disease. It has been proven that asparagus polysaccharide, one of the most active derivates from the traditional medicine asparagus, possesses notable antitumor properties. However, little is known about the efficacy of asparagus polysaccharide as an adjuvant for liver cancer chemotherapy. Herein, we reported that asparagus polysaccharide and its embolic agent form, asparagus gum, significantly inhibited liver tumor growth with transcatheter arterial chemoembolization (TACE) therapy in an orthotopic hepatocellular carcinoma (HCC) tumor model, while significantly inhibiting angiogenesis and promoting tumor cell apoptosis. Moreover, asparagine gelatinous possessed immunomodulatory functions and showed little toxicity to the host. These results highlight the chemotherapeutic potential of asparagus polysaccharide and warrant a future focus on development as novel chemotherapeutic agent for liver cancer TACE therapy.

[Destruction of Walker-256 tumor vasculature by a novel ultrasound cavitation technique].

OBJECTIVE: To explore the feasibility of disrupting tumor microcirculation by the cavitation of microbubbles enhanced ultrasound (US) and analyze its pathological mechanism. METHODS: Twenty-four SD male rats with subcutaneously transplanted Walker-256 tumor were divided into 3 groups, i.e. ultrasound plus microbubbles group (US + MB), US group and sham group. Pulsed US was delivered to tumor for 3 minutes during an intravenous infusion of microbubbles at 0.2 ml/kg in the US + MB group. The control groups received only the US exposure or the MB injection. Tumor perfusion was visualized with contrast enhanced ultrasound before and 0 min after treatment. Finally the pathological examination was performed. RESULTS: The contrast perfusion of Walker-256 tumors vanished immediately after treatment in the US + MB group and the gray scale value (GSV) decreased from 121 ± 12 (pre-treatment) to 81 ± 9 (post-treatment, P < 0.01). There was no significant difference of GSV before and after treatment in two control groups (P > 0.05). The GSV values were 112 ± 14 and 111 ± 12 pre-treatment and 113 ± 14 and 103 ± 13 post-treatment in the sham and US groups. The pathological examination showed remarkable hemorrhage, endothelial injuries, increased intercellular edema and in situ thrombosis. CONCLUSION: Microbubble-enhanced ultrasound can significantly disrupt tumor vasculature and block its circulation. And it may become a novel physical anti-angiogenetic therapy for tumor.

Fingolimod potentiates the effects of sunitinib malate in a rat breast cancer model.

Most of the antiangiogenic strategies used in oncology principally target endothelial cells through the vascular endothelial growth factor (VEGF) pathway. Multiple kinase inhibitors can secondarily reduce mural cell stabilization of the vessels by blocking platelet-derived growth factor receptor (PDGFR) activity. However, sphingosine-1-phosphate (S1P), which is also implicated in mural cell recruitment, has yet to be targeted in clinical practice. We therefore investigated the potential of a simultaneous blockade of the PDGF and S1P pathways on the chemotactic responses of vascular smooth muscle cells (VSMCs) and the resulting effects of this blockade on breast tumor growth. Due to crosstalk between the S1P and PDGF pathways, we used AG1296 and/or VPC-23019 to inhibit PDGFR-ß and S1PR1/S1PR3 receptors, respectively. We showed that S1PR1 and S1PR3 are the principal receptors that mediate the S1P chemotactic signal on rat VSMCs and that they act synergistically with PDGFR-ß during PDGF-B signaling. We also showed that simultaneous blockade of the PDGFR-ß and S1PR1/S1PR3 signals had a synergistic effect, decreasing VSMC migration velocity toward endothelial cell and breast carcinoma cell-secreted cytokines by 65-90%. This blockade also strongly decreased the ability of VSMCs to form a three-dimensional cell network. Similar results were obtained with the combination of sunitinib malate (a VEGFR/PDGFR kinase inhibitor) and fingolimod (an S1P analog). Sunitinib malate is a clinically approved cancer treatment, whereas fingolimod is currently indicated only for treatment of multiple sclerosis. Orally administered, the combination of these drugs greatly decreased rat breast tumor growth in a syngeneic cancer model (Walker 256). This bi-therapy did not exert cumulative toxicity and histological analysis of the tumors revealed normalization of the tumor vasculature. The simultaneous blockade of these signaling pathways with sunitinib malate and fingolimod may provide an effective means of reducing tumor angiogenesis, and may improve the delivery of other chemotherapies.

Radiopharmaceutical tracking of particles injected into tumors: a model to study clearance kinetics.

OBJECTIVES: Success of selective drug therapies depends on the drug's depot time in the target to treat. Depot time is currently being prolonged, using drug-eluting beads or microspheres for selective internal radiation therapy. The purpose of this study was to establish a model for investigating the depot time of particles injected into tumors in relation to tumor vascularization and particle size. MATERIALS AND METHODS: An animal model with two different vascularized tumors (Walker Carcinoma 256 (hypervascularized) and Yoshida Sarcoma (hypovascularized)) was used. The tumors were implanted into the hind leg of Wistar rats. When the tumors reached 10-15mm, rhenium radiolabeled particles of 25microm and 0.3microm were percutaneously injected into the tumors: large particles (Re-188 microspheres) in 10 hypo- and 10 hypervascularized tumors and small particles (Re-186 sulfid colloid) in 4 hypo- and 16 hypervascularized tumors with the co-injection of the vasoconstrictor, adrenalin (0.01 mg), into 8 hypervascularized tumors. Tumor activity was measured with a gamma camera at 10 min, 1h, 3h, 6h, 14h and 48h p.i. In addition, activity in the lung, liver, spleen, kidneys, and lymph nodes was measured at 48 h p.i. Measurements were adjusted for decay times and then compared. RESULTS: Drainage of the injected particles is bi-phasic, characterized by a fast wash-out. At 10 min p.i., intratumoral activity decreases to 70% of the initially injected activity. This is followed by a slow decline at 48 h p.i in which intratumoral activity decreases to at least 60% of the initially injected activity. Slow decline is independent of particle size and vascularization, whereas fast leakage depends on both. Co-injecting adrenalin significantly reduced the wash-out of the small particles. Radiolabeled microspheres accumulated mainly in the lungs, smaller colloids in the liver. CONCLUSIONS: Particle stay time, biodistribution, and stability can be easily monitored as shown in this animal model. The hematogeneous wash-out can be reduced, using larger particles and vasoconstrictors. Prolonged retention is independent of vascularization and particle size and appears to be sufficient for therapy.

Specific thermographic changes during Walker 256 carcinoma development: differential infrared imaging of tumour, inflammation and haematoma.

BACKGROUND: Infrared imaging measures spatial variations in the skin temperature aiming to determine pathological processes; hence possible use of this non-invasive analytical method in cancer detection is emerging. METHODS: Infrared thermal imaging was used to detect changes in rat skin surface temperature associated with experimental cancer development (Walker 256 carcinoma), inflammation (upon s.c. Sephadex injection) and haematoma (provoked by s.c. blood coagulate injection). Infrared camera with a geometric resolution of 76,800 pixels, spectral range of 8-14 microm and the minimal detectable temperature resolution of 0.07 degrees C with spatial resolution of 0.48 mm at measuring distance of 30 cm was used to obtain computerised thermal scans. Genuine ThermoWEB software developed for remote internet control as open source software was used. RESULTS: The raise of peripheral temperature was observed after induction of local inflammation or haematoma. Opposite to that, transient decrease of the skin surface temperature was observed after tumour transplantation. Progressive growth of tumour was associated with the raise of the skin surface temperature from the 10th day after tumour inoculation, when the tumours developed supportive neoangiogenic blood supply, as verified by histology. CONCLUSION: While the raise of peripheral temperature in advanced tumour was caused by neoangiogenesis, the reduction in skin surface temperature in an early period after tumour cell inoculation indicated a decay of transplanted tumour cells due to the immune response and the lack of blood supply. Thus, infrared thermal imaging may have considerable value in evaluation of the tumour development and discrimination of cancer from inflammation and haematoma.

The effect of electromagnetic field and local inductive hyperthermia on nonlinear dynamics of the growth of transplanted animal tumors.

AIM: To examine the effects of electromagnetic field with amplified magnetic component and local inductive hyperthermia (IH) on nonlinear dynamics of the growth of animal tumors. MATERIALS AND METHODS: Guerin carcinoma, Lewis lung carcinoma, sarcoma 45, Walker 256 carcinosarcoma and Pliss lymphosarcoma were studied. The animal tumors were exposed inside of loop aerial, 3 cm in diameter locally for 30 min. Parameters of electromagnetic irradiation (EI): frequency 40 MHz, magnetic intensity 72 A/m, electric intensity 200 V/m and the output power 50 W. The temperature measured by immersion of thermocouple inside the center of the tumor didn't exceed 38.5-39.5 degrees C. Nonlinear dynamics of the growth of animal tumors was analyzed by autocatalytic equation. The heterogeneity of ultrasonic image of the tumor was analyzed by Moran spatial autocorrelation. RESULTS: The strongest inhibition effect under the influence of EI was in Pliss lymphosarcoma and sarcoma 45. The growth stimulation of animal tumors after EI was recorded in Walker 256 carcinosarcoma. The use of mild IH increased the blood flow in the tumor of Guerin carcinoma. CONCLUSION: These results are important for clinical application because they testify the necessity of optimization of schemes for local EI during anticancer neoadjuvant therapy with the use of drugs or magnetic nanoparticles. The use of mild IH as a basis for the monotherapy of malignant tumors is not expedient.

[An experimental study of energy controllable steep pulse in the treatment of rat with subcutaneous transplantive tumor].

This experimental study was designed to investigate the effects and the expressions of microvessel density (MVD), vascular endothelial growth factor (VEGF) on the transplanted tumor in the rat model with Walker-256 after energy controllable steep pulse(ECSP). The experiment revealed that the steep pulse electrical field has better effect on tumor, compared with the control. The positive cell staining intensity of VEGF in the control group was significantly higher than that in ECSP group (P < 0.05). The number of MVD in the tumor tissues of ECSP group was significantly lower than that of tumor control group (P < 0.05). These results showed that ECSP could inhibit the growth and angiogenesis of tumor and its pathway is to down-regulate the expression of VEGF possibly.

Host stromal bradykinin B2 receptor signaling facilitates tumor-associated angiogenesis and tumor growth.

We evaluated the significance of the host kallikrein-kinin system in tumor angiogenesis and tumor growth using two rodent models genetically deficient in a kallikrein-kinin system. Inoculation of Walker 256 carcinoma cells into the s.c. tissues of the back of normal Brown Norway Kitasato rats (BN-Ki rats) resulted in the rapid development of solid tumors with marked angiogenesis. By contrast, in kininogen-deficient Brown Norway Katholiek rats (BN-Ka rats), which cannot generate intrinsic bradykinin (BK), the weights of the tumors and the extent of angiogenesis were significantly less than those in BN-Ki rats. Daily administration of B(2) receptor antagonists significantly reduced angiogenesis and tumor weights in BN-Ki rats to levels similar to those in BN-Ka rats but did not do so in BN-Ka rats. Angiogenesis and tumor growth were significantly suppressed in B(2) receptor knockout mice bearing sarcoma 180 compared with their wild-type counterparts. Immunoreactive vascular endothelial growth factor (VEGF) was localized in Walker tumor stroma more extensively in BN-Ki rats than in BN-Ka rats, although immunoreactive B(2) receptor also was detected in the stroma to the same extent in both types of rats. Cultured stromal fibroblasts isolated from BN-Ki rats and BN-Ka rats produced VEGF in response to BK (10(-8)-10(-6) m), and this stimulatory effect of BK was abolished with a B(2) receptor antagonist, Hoe140 (10(-5) m). These results suggest that BK generated from kininogens supplied from the host may facilitate tumor-associated angiogenesis and tumor growth by stimulating stromal B(2) signaling to up-regulate VEGF production mainly in fibroblasts.

Transient increased expression of VEGF and MMP-1 in a rat liver tumor model after hepatic arterial occlusion.

BACKGROUND/AIMS: This study investigates the influence of hepatic arterial occlusion (HAO) on blood perfusion of transplanted cancer in rat's liver, and the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-1 (MMP-1) and explores the mechanisms involved in transcatheter arterial embolization (TAE)-induced metastasis of liver cancer preliminarily. METHODOLOGY: Walker 256 carcinosarcoma was transplanted into rat's liver to create the liver cancer model. Hepatic arterial ligation (HAL) was used to block the hepatic arterial blood supply and simulate TAE. Rats bearing tumor were divided into three groups: control, laparotomy control, and HAL groups. Blood perfusion of tumor was analyzed by a Hoechst 33342 labeling assay. The level of serum VEGF was assayed by ELISA; and the expression of VEGF and MMP-1 mRNA was detected by in situ hybridization. RESULTS: Two days after HAL, the number of Hoechst 33342 labeled cells which represent the blood perfusion of the tumor directly and hypoxia of tumor indirectly in the HAL group decreased significantly compared with that in the control group (329.1+/-29.3 vs. 383.6+/-19.2, P<0.01). The level of serum VEGF in the HAL group increased significantly compared with that of the control group (92.5+/-43.9 pg/mL vs. 54.9+/-19.3 pg/mL, P<0.05). The expression of VEGF and MMP-1 mRNA in the tumor tissue of the HAL group increased significantly compared with that of the control and the laparotomy control groups (P<0.05). The blood perfusion data of the tumor, represented by number of Hoechst 33342 labeled cells, showed an inverse correlation with the expression of VEGF mRNA in tumor tissue (P<0.05). While 6 days after HAL, the blood perfusion of tumor in HAL group decreased and the expression of VEGF and MMP-1 increased only slightly, not significantly, compared with that in the control group. CONCLUSIONS: In conclusion, blockage of hepatic arterial blood supply results in transient decreased blood perfusion and increased expression of metastasis-associated genes VEGF and MMP-1 of transplanted liver cancer in rats. Decreased blood perfusion and hypoxia may be the major reason for up-regulated expression of VEGF. Better understanding of the mechanisms involved with TAE-induced metastasis may lead to the enhancement of the long-term effects of TAE for liver cancer.

Disposition of WR-1065 in the liver of tumor-bearing rats following regional vs systemic administration of amifostine.

PURPOSE: Amifostine is a prodrug in which selectivity is largely determined by the preferential formation and uptake of its cytoprotective metabolite, WR-1065, in normal tissues as a result of differences in membrane-bound alkaline phosphatase activity. It was hypothesized that amifostine may be a good candidate for regional drug delivery to the liver because of its large hepatic extraction and total body clearance. METHODS: Rat livers were implanted with Walker-256 tumors. The tumor-bearing rats received 15 min infusions of amifostine (200 mg/kg) via the portal vein or the femoral vein. WR-1065 concentrations in the blood, liver and tumor were measured at various times. RESULTS: The WR-1065 tumor portal dosing AUC15-60 was 40% of systemic dosing, and tumor concentrations following portal dosing were one-fifth of that following systemic dosing. The portal dosing WR-1065 liver AUC15-60 was 60% higher than the values for systemic dosing. The liver/tumor concentration ratios of WR-1065 following portal dosing were up to 8-fold higher than the ratio following systemic administration. Unfortunately, systemic exposure to WR-1065 was greater following portal vs systemic amifostine. CONCLUSIONS: Amifostine may provide increased liver protection and decreased tumor protection from radio- or chemotherapy when administered by the portal vein. However, portal dosing also increases systemic exposure to WR-1065, which is associated with hypotension.

Influence of transarterial chemoembolization on angiogenesis and expression of vascular endothelial growth factor and basic fibroblast growth factor in rat with Walker-256 transplanted hepatoma: an experimental study.

AIM: After transarterial chemoembolization (TACE), the residual cancer cells are under extensive hypoxic or even anoxic environment. Hypoxia can lead to adaptive responses. For example, angiogenesis will help these cells survive. In this study, we examined the effect of TACE on angiogenesis and expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) and to assess their relevance to Walker-256 transplanted hepatoma. METHODS: Male Wistar rats were inoculated with Walker-256 tumor in the left lobe of liver. Angiography and transarterial chemoembolization were performed at d14 after transplantation. Sixty rats bearing walker-256 transplanted hepatoma were randomly divided into control group, arterial infusion group and TACE group. Each group consisted of twenty rats. Normal saline, 5-Fu, 5-Fu and lipiodol were infused through hepatic artery respectively. Two weeks after the infusion, staining of factor VIII, VEGF and b-FGF was performed by immunohistochemistry method in routine paraffin-embedded sections. Microvessel density (MVD) was counted in endothelial cells with positive factor VIII. Their expression levels were analyzed in conjunction with the pathologic features. RESULTS: While a smaller tumor volume was found in TACE group (F=37.818, P<0.001), no statistical differences between MVD and expression of VEGF and b-FGF were found among the 3 groups. MVD of the control group, chemotherapy group and chemoemoblization group was 80.84+/-24.24, 83.05+/-20.29 and 85.20+/-23.91 (F=0.193, P=0.873), respectively. The positive expression of VEGF and b-FGF was 75%, 75%, 85% (chi2=0.449, P=0.799) and 30%, 25%, 30% (chi2=0.141, P=0.922), respectively. Statistical analysis revealed a positive correlation between the expression of VEGF and MVD (r=0.552, P<0.001). CONCLUSION: There has been little influence of lipiodol chemoembolization on the formation of tumor angiogenesis, but the development of neovascularization and expression of VEGF play important roles in establishment of collateral circulation and reconstruction of blood supply of residual cancer tissue.

Influence of hepatic arterial blockage on blood perfusion and VEGF, MMP-1 expression of implanted liver cancer in rats.

AIM: To investigate the influence of hepatic arterial blockage on blood perfusion of transplanted cancer in rat liver and the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-1 (MMP-1), and to explore the mechanisms involved in transarterial embolization (TAE)-induced metastasis of liver cancer preliminarily. METHODS: Walker 256 carcinosarcoma was transplanted into rat liver to establish the liver cancer model. Hepatic arterial ligation (HAL) was used to block the hepatic arterial blood supply and simulate TAE. Blood perfusion of tumor in control, laparotomy control, and HAL group was analyzed by Hoechst 33342 labeling assay, the serum VEGF level was assayed by ELISA, the expression of VEGF and MMP-1 mRNA was detected by in situ hybridization. RESULTS: Two days after HAL, the number of Hoechst 33342 labeled cells which represent the blood perfusion of tumor directly and hypoxia of tumor indirectly in HAL group decreased significantly compared with that in control group (329+/-29 vs 384+/-19, P<0.01). The serum VEGF level in the HAL group increased significantly as against that of the control group (93 ng.L(-1)+/-44 ng.L(-1) vs 55 ng.L(-1)+/-19 ng.L(-1), P<0.05). The expression of VEGF and MMP-1 mRNA in the tumor tissue of the HAL group increased significantly compared with that of the control and the laparotomy control groups (P<0.05). The blood perfusion data of the tumor, represented by the number of Hoechst 33342 labeled cells, showed a good linear inverse correlation with the serum VEGF level (r=-0.606, P<0.05) and the expression of VEGF mRNA in the tumor tissue ( r =-0.338, P<0.01). CONCLUSION: Blockage of hepatic arterial blood supply results in decreased blood perfusion and increased expression of metastasis-associated genes VEGF and MMP-1 of transplanted liver cancer in rats. Decreased blood perfusion and hypoxia may be the major cause of up-regulated expression of VEGF.

Technetium-99m HL91 uptake as a tumour hypoxia marker: relationship to tumour blood flow.

Technetium-99m HL91 (HL91) is a potential agent for imaging hypoxic tissue in vivo. To elucidate the relationship between hypoxia and blood flow in a tumour, dual-tracer autoradiography with HL91 and carbon-14 iodoantipyrine (IAP) was performed in a tumour-bearing rat model. The distribution of each tracer was analysed visually and semiquantitatively. In the tumours with central necrotic areas, HL91 uptake was marked around the necrotic areas whereas IAP uptake was marked at the periphery of the tumours, around the areas of marked HL91 uptake. Normalized HL91 uptake (%HL91) was highest in the low normalized IAP uptake (%IAP) fraction in the non-necrotic areas. There was a weak negative correlation between %HL91 and %IAP in the non-necrotic areas (r = -0.322, P<0.0001). In tumours with few or no necrotic areas, HL91 uptake was heterogeneous throughout the tumours, while IAP uptake predominantly occurred at the periphery of the tumours. %HL91 was higher in the inner two-thirds of the tumour than in the outer third. There was again a weak negative correlation between %HL91 and %IAP (r = -0.354, P<0.0001). This study confirmed that high HL91 uptake is related to low blood flow. The marked HL91 uptake around the necrotic region suggests the presence of chronic hypoxia in a tumour.

Inhibition of tumor growth and microvascular angiogenesis by the potent angiogenesis inhibitor, TNP-470, in rats.

The antiangiogenic effects of TNP-470 on the neovascularization of tumors were studied by examining ultrastructural alterations in the vasculature and interstitial fluid pressure (IFP) of tumors. Wistar rats were first inoculated subcutaneously (s.c.) with the Walker 256 carcinosarcoma cell line, then either vehicle medium or TNP-470, 30 mg/kg, was injected s.c. on day 1. A tumor growth assay, the necrotic area, and the IFP in the tumor were all measured on day 12. The antiangiogenic effects of TNP-470 were studied by scanning electron microscopic images of tumor vascular casts. TNP-470 was observed to inhibit tumor growth and increase the necrotic area significantly. In the TNP-470-treated group, the IFP in the superficial layer, defined as 2-3 mm from the tumor capsule, and in the deep layer, defined as 8-10 mm from the tumor capsule, were significantly higher than the corresponding values in the control. Moreover, vascular casts showed a significant reduction in the budding of sprouts in the superficial layer, and a decrease in the maximum diameter of the tumor vessels in the deep layer. It is possible that the higher IFP in the TNP-470-treated tumors might have prevented tumor vessel dilation. The findings of this study demonstrated that TNP-470 inhibited the budding of tumor vessel sprouts, and increased the IFP. These processes seem to act synergistically to suppress tumor angiogenesis.

Measurement of tumor vascular volume and mean microvascular random flow velocity magnitude by dynamic Gd-DTPA-albumin enhanced and diffusion-weighted MRI.

Tumor vascular volume fraction and the magnitude of the mean microvascular random flow velocity were measured in an animal tumor model by combining dynamic Gd-DTPA-albumin enhanced MRI and diffusion-weighted MRI in conjunction with a compartmental modeling analysis. The vascular volume fraction maps were obtained from the dynamic Gd-DTPA-albumin enhanced MRI measurement. It was found that the vascular volume fraction for Walker 256 tumor was higher within the outgrowing rim and decreased towards the central region. The average value obtained from five animals was 0.062 +/- 0.009 ml/g. By using the vascular volume fraction from the Gd-DTPA-albumin enhanced MRI measurement, maps of the magnitude of the mean microvascular random flow velocity were obtained from the diffusion-weighted MRI measurements with the compartmental modeling analysis. The relative extravascular and intravascular contributions to the diffusion-weighted MRI signal were determined for three tissue groups with different Gd-DTPA-albumin enhancement characteristics, and the flow and molecular diffusion-induced attenuation factors for the intravascular compartment were also compared. The mean microvascular random flow velocity magnitude maps were obtained with an average value of 0.67 +/- 0.06 mm/s.