Three-dimensional imaging of tumor angiogenesis

OBJECTIVE: To three-dimensionally visualize the microvessel environment of tumor angiogenesis by confocal laser scanning microscopy (CLSM). STUDY DESIGN: To reveal underlying mechanisms of tumor angiogenesis, a 7, 12-dimethylbenz(a) anthracene-induced rat cancer model was used. For demonstrating tumor vasculature, fluorescence injection method (FITC-conjugated gelatin solution) was employed. FITC gelatin was injected into the left ventricle of the rat heart. After complete perfusion, the mammary glands were resected, fixed under ice cold conditions and subjected to immunohistochemistry (IHC) for tumor cells. The LSM-410 (Carl Zeiss, Jena, Germany) was employed on thick sections (300-2,000 microns) to elucidate detailed microvessel networks (MVN) and tumor cells. RESULTS: Tumor vasculature on thick sections was clearly detected by CLSM at the maximum focus depth of 2,000 microns. Three-dimensional (3-D), reconstructed images of normal mammary glands showed regular and linear MVN. In DMBA-induced mammary cancer, vascular density of MVN was markedly increased and showed an anastomosing, irregular MVN pattern. Furthermore, focal segmentation and tortuous, branching patterns of microvessels were also seen. CONCLUSION: Application of the fluorescence injection method and IHC using CLSM was very useful for studying the 3-D relationship between tumor angiogenesis and neoplastic epithelial changes. These results suggest that application of this technique is ideal for studying 3-D imaging of tumor angiogenesis.     

TNF-alpha downregulates vascular endothelial Flk-1 expression in human melanoma xenograft model

High-dose TNF with melphalan has significant antitumor activity in regional perfusion of the limbs and liver in human malignancies. TNF is believed to target tumor vasculature, but the precise molecular mechanism is unknown. The present study demonstrates that TNF downregulates the VEGF receptor, fetal liver kinase-1 (Flk-1), on tumor endothelium in a human melanoma xenograft model. NIH1286 human melanoma cells were transduced with a 720-bp fragment of the human VEGF(121) gene to develop well-vascularized tumors that served as an amplified system for measuring Flk-1 expression changes. We injected 5 x 10(6) cells subcutaneously, each of two distinct single cell clones (NIH1286/3 and NIH1286/15), into athymic nude mice to produce tumors approximately 10 mm in size. Each animal then received either BSA or TNF in BSA by tail vein. Tumors harvested at different time points post-TNF were analyzed for Flk-1 mRNA and protein expression. Data obtained showed that intravascular TNF downregulated Flk-1 expression in tumor endothelial cells. This effect could contribute to the antitumor activity of TNF known to target tumor vasculature.     

Transplantation of placenta‐derived mesenchymal stem cells enhances angiogenesis after ischemic limb injury in mice

Mesenchymal stem cell‐based therapy has emerged as a promising approach for the treatment of peripheral arterial disease. The purpose of this study was to examine the potential effects of human placenta‐derived mesenchymal stem cells (PMSCs) on mouse hindlimb ischemia. PMSCs were isolated from human placenta tissue and characterized by flow cytometry. An in vivo surgical ligation‐induced murine limb ischemia model was generated with fluorescent dye (CM‐DiI) labelled PMSCs delivered via intramuscular injection. Our data show that PMSCs treatment significantly enhanced microvessel density, improved blood perfusion and diminished pathologies in ischemic mouse hindlimbs as compared to those in the control group. Further immunostaining studies suggested that injected PMSCs can incorporate into the vasculature and differentiate into endothelial and smooth muscle cells to enhance angiogenesis in ischemic hind limbs. This may in part explain the beneficial effects of PMSCs treatment. Taken together, we found that PMSCs treatment might be an effective treatment modality for treatment of ischemia‐induced injury to mouse hind limbs by enhancement of angiogenesis.     

Tubulin-Destabilizing Agent BPR0L075 Induces Vascular-Disruption in Human Breast Cancer Mammary Fat Pad Xenografts

BPR0L075, 6-methoxy-3-(3',4',5'-trimethoxy-benzoyl)-1H-indole, is a tubulin-binding agent that inhibits tubulin polymerization by binding to the colchicine-binding site. BPR0L075 has shown antimitotic and antiangiogenic activity in vitro. The current study evaluated the vascular-disrupting activity of BPR0L075 in human breast cancer mammary fat pad xenografts using dynamic bioluminescence imaging. A single dose of BPR0L075 (50 mg/kg, intraperitoneally (i.p.)) induced rapid, temporary tumor vascular shutdown (at 2, 4, and 6 hours); evidenced by rapid and reproducible decrease of light emission from luciferase-expressing orthotopic MCF7 and MDA-MB-231 breast tumors after administration of luciferin substrate. A time-dependent reduction of tumor perfusion after BPR0L075 treatment was confirmed by immunohistological staining of the perfusion marker Hoechst 33342 and tumor vasculature marker CD31. The vasculature showed distinct recovery within 24 hours post therapy. A single i.p. injection of 50 mg/kg of BPR0L075 initially produced plasma concentrations in the micromolar range within 6 hours, but subsequent drug distribution and elimination caused BPR0L075 plasma levels to drop rapidly into the nanomolar range within 24 h. Tests with human umbilical vein endothelial (HUVEC) cells and tumor cells in culture showed that BPR0L075 was cytotoxic to both tumor cells and proliferating endothelial cells, and disrupted pre-established vessels in vitro and ex vivo. In conclusion, BPR0L075 caused rapid, albeit, temporary tumor vascular shutdown and led to reduction of tumor perfusion in orthotopic human breast cancer xenografts, suggesting that this antimitotic agent may be useful as a vascular-disrupting cancer therapy.     

Vascular remodeling marks tumors that recur during chronic suppression of angiogenesis

The potential for avoiding acquired resistance to therapy has been proposed as one compelling theoretical advantage of antiangiogenic therapy based on the normal genetic status of the target vasculature. However, previous work has demonstrated that tumors may resume growth after initial inhibition if antiangiogenic blockade is continued for an extended period. The mechanisms of this recurrent growth are unclear. In these studies, we characterized molecular changes in vasculature during apparent resumption of xenograft growth after initial inhibition by vascular endothelial growth factor blockade, "metronome" topotecan chemotherapy, and combined agents in a xenograft murine model of human Wilms' tumor. Tumors that grew during antiangiogenic blockade developed as viable clusters surrounding strikingly remodeled vessels. These vessels displayed significant increases in diameter and active proliferation of vascular mural cells and expressed platelet-derived growth factor-B, a factor that functions to enhance vascular integrity via stromal cell recruitment. In addition, remodeled vessels were marked by expression of ephrinB2, required for proper assembly of stromal cells into vasculature. Thus, enhanced vascular stability appears to characterize tumor vessel response to chronic antiangiogenesis, features that potentially support increased perfusion and recurrent tumor growth.     

Tumor necrosis factor-α-activated mesenchymal stem cells promote endothelial progenitor cell homing and angiogenesis

Mesenchymal stem cells (MSCs) accelerate regeneration of ischemic or injured tissues by stimulation of angiogenesis through a paracrine mechanism. Tumor necrosis factor-α (TNF-α)-activated MSCs secrete pro-angiogenic cytokines, including IL-6 and IL-8. In the present study, using an ischemic hindlimb animal model, we explored the role of IL-6 and IL-8 in the paracrine stimulation of angiogenesis and tissue regeneration by TNF-α-activated MSCs. Intramuscular injection of conditioned medium derived from TNF-α-treated MSCs (TNF-α CM) into the ischemic hindlimb resulted in attenuated severe limb loss and stimulated blood perfusion and angiogenesis in the ischemic limb. Immunodepletion of IL-6 and IL-8 resulted in attenuated TNF-α CM-stimulated tissue repair, blood perfusion, and angiogenesis. In addition, TNF-α CM induced migration of human cord blood-derived endothelial progenitor cells (EPCs) through IL-6- and IL-8-dependent mechanisms in vitro. Intramuscular injection of TNF-α CM into the ischemic limb led to augmented homing of tail vein-injected EPCs into the ischemic limb in vivo and immunodepletion of IL-6 or IL-8 from TNF-α CM attenuated TNF-α CM-stimulated homing of EPCs. In addition, intramuscular injection of recombinant IL-6 and IL-8 proteins resulted in increased homing of intravenously transplanted EPCs into the ischemic limb and improved blood perfusion in vivo. These results suggest that TNF-α CM stimulates angiogenesis and tissue repair through an increase in homing of EPCs through paracrine mechanisms involving IL-6 and IL-8.     

Therapeutic angiogenesis by ex vivo expanded erythroid progenitor cells

Recent reports have demonstrated that erythroid progenitor cells contain and secrete various angiogenic cytokines. Here, the impact of erythroid colony-forming cell (ECFC) implantation on therapeutic angiogenesis was investigated in murine models of hindlimb ischemia. During the in vitro differentiation, vascular endothelial growth factor (VEGF) secretion by ECFCs was observed from day 3 (burst-forming unit erythroid cells) to day 10 (erythroblasts). ECFCs from day 5 to day 7 (colony-forming unit erythroid cells) showed the highest VEGF productivity, and day 6 ECFCs were used for the experiments. ECFCs contained larger amounts of VEGF and fibroblast growth factor-2 (FGF-2) than peripheral blood mononuclear cells (PBMNCs). In tubule formation assays with human umbilical vein endothelial cells, ECFCs stimulated 1.5-fold more capillary growth than PBMNCs, and this effect was suppressed by antibodies against VEGF and FGF-2. Using an immunodeficient hindlimb ischemia model and laser-Doppler imaging, we evaluated the limb salvage rate and blood perfusion after intramuscular implantation of ECFCs. ECFC implantation increased both the salvage rate (38% vs. 0%, P < 0.05) and the blood perfusion (82.8% vs. 65.6%, P < 0.01). In addition, ECFCs implantation also significantly increased capillaries with recruitment of vascular smooth muscle cells and the capillary density was 1.6-fold higher than in the control group. Continuous production of human VEGF from ECFCs in the skeletal muscle was confirmed at least 7 days after the implantation. Implantation of ECFCs promoted angiogenesis in ischemic limbs by supplying angiogenic cytokines (VEGF and FGF-2), suggesting a possible novel strategy for therapeutic angiogenesis.     

Canstatin, a novel matrix-derived inhibitor of angiogenesis and tumor growth

We isolated and identified an endogenous 24-kDa human basement membrane-derived inhibitor of angiogenesis and tumor growth, termed canstatin. Canstatin, a fragment of the alpha2 chain of type IV collagen, was produced as a recombinant molecule in Escherichia coli and 293 embryonic kidneys cells. Canstatin significantly inhibited human endothelial cell migration and murine endothelial cell tube formation. Additionally, canstatin potently inhibited 10% fetal bovine serum-stimulated endothelial cell proliferation and induced apoptosis, with no inhibition of proliferation or apoptosis observed on non-endothelial cells. Inhibition of endothelial proliferation was not concomitant with a change in extracellular signal-regulated kinase activation. We demonstrate that apoptosis induced by canstatin was associated with a down-regulation of the anti-apoptotic protein, FLIP. Canstatin also suppressed in vivo growth of large and small size tumors in two human xenograft mouse models with histology revealing decreased CD31-positive vasculature. Collectively, these results suggest that canstatin is a powerful therapeutic molecule for suppressing angiogenesis.     

Dual function of RGD-modified VEGI-192 for breast cancer treatment

Identification of endogenous angiogenesis inhibitors has led to development of an increasingly attractive strategy for cancer therapy and other angiogenesis-driven diseases. Vascular endothelial growth inhibitor (VEGI), a potent and relatively nontoxic endogenous angiogenesis inhibitor, has been intensively studied, and this work shed new light on developing promising anti-angiogenic strategies. It is well-documented that the RGD (Arg-Gly-Asp) motif exhibits high binding affinity to integrin α(v)β(3), which is abundantly expressed in cancer cells and specifically associated with angiogenesis on tumors. Here, we designed a fusion protein containing the special RGD-4C motif sequence and VEGI-192, aimed at offering more effective multiple targeting to tumor cells and tumor vasculature, and higher anti-angiogenic and antitumor efficacy. Functional tests demonstrated that the purified recombinant human RGD-VEGI-192 protein (rhRGD-VEGI-192) potently inhibited endothelial growth in vitro and suppressed neovascularization in chicken chorioallantoic membrane in vivo, to a higher degree as compared with rhVEGI-192 protein. More importantly, rhRGD-VEGI-192, but not rhVEGI-192 protein, could potentially target MDA-MB-435 breast tumor cells, significantly inhibiting growth of MDA-MB-435 cells in vitro, triggered apoptosis in MDA-MB-435 cells by activation of caspase-8 as well as caspase-3, which was mediated by activating the JNK signaling associated with upregulation of pro-apoptotic protein Puma, and consequently led to the observed significant antitumor effect in vivo against a human breast cancer xenograft. Our study indicated that the RGD-VEGI-192 fusion protein might represent a novel anti-angiogenic and antitumor strategy.     

Delta4, an endothelial specific Notch ligand expressed at sites of physiological and tumor angiogenesis

Delta-Notch signalling regulates cell-fate choices in a variety of tissues during development. We report the expression of Delta4 (D14) in arterial endothelium during mouse embryogenesis and in the endothelium of tumor blood vessels. The expression of D14 in the mouse begins at 8 dpc in the dorsal aortae, umbilical artery and the heart. Subsequent expression is restricted to smaller vessels and capillaries and is reduced in most adult tissues. However, it is high in the vasculature of xenograft human tumors in the mouse, in endogenous human tumors and is regulated by hypoxia. These data implicate D14 and the Notch signalling pathway in angiogenesis and suggest possible new targets for antiangiogenic tumor therapy.     

COX-2 Inhibition Combined with Radiation Reduces Orthotopic Glioma Outgrowth by Targeting the Tumor Vasculature

Cyclooxygenase 2 (COX-2) inhibitors have been shown to enhance tumor''s response to radiation in several animal models. The strong association of COX-2 and angiogenesis suggests that the tumor vasculature may be involved in this process. The current study investigated whether treatment with the COX-2 inhibitor E-6087 could influence response to local radiation in orthotopically growing murine gliomas and aimed to analyze the involvement of the tumor vasculature. GL261 glioma cells were injected into the cerebrum of C57bl/6 mice. From day 7 after tumor cell injection, mice were treated with COX-2 inhibitor at 50 mg/kg i.p. every third day. Radiation consisted of three fractions of 2 Gy given daily from day 9 to day 11. Mice were killed at day 21. The COX-2 inhibitor significantly enhanced the response to radiation, reducing mean volume to 32% of tumors treated with radiation only. The combination treatment neither increased apoptosis of tumor cells or stromal cells nor affected tumor microvascular density. In vitro, E-6087 and its active metabolite did not affect clonogenic survival of GL261 cells or human umbilical vein endothelial cell after radiation. In vivo, however, there was a nonsignificant increase in Angiopoietin (Ang)-1 and Tie-2 mRNA levels and a decrease of Ang-2 mRNA levels after combination treatment. These changes coincided with a significant increase in α-smooth muscle actin-positive pericyte coverage of tumor vessels. In conclusion, the antitumor effect of radiation on murine intracranial glioma growth is augmented by combining with COX-2 inhibition. Our findings suggest an involvement of the tumor vasculature in the observed effects.     

Immunological and biological identification of tumour necrosis-like factor in sponges: endotoxin that mediates necrosis formation in xenografts.

Xenografts of the sponge Geodia cydonium in its closely related species G. rovinjensis resulted in a rapid rejection of the graft within a period of 5 days. We identified an immunoreactive tumour necrosis factor (TNF)-like activity in the xenograft (Mr of 30,000) two days after grafting. In-vivo injection of 5 micrograms human recombinant TNF-alpha induced cytotoxicity in sponge cells in the same pattern and time course as during natural xenograft rejection. Anti-TNF-alpha polyclonals were found to react with xenograft extracts, by Western blot analysis, as from day 2 after grafting. Using ELISA we detected the TNF-like activity from day 2 after grafting with peak levels at days 4 and 5, where the amount was 0.72 ng/micrograms tissue DNA. By day 1, gp27 (inhibitory aggregation factor) is already formed in the xenograft. In-vitro experiments on isolated G. cydonium cells showed that addition of purified gp27 induced the production of the TNF-like activity (up to 13.5 ng/ml). Evidence is presented that gp27 is a product of the gp180 lectin receptor. We conclude that gp27 induces TNF-like factor production, resulting in destruction and dissolution of the xenograft after 5 days.     

Recruitment of mononuclear leucocytes to osteoarthritic human synovial xenografts in the ears of SCID mice

A system has been established to assess the recruitment of 99mTc-hexamethylpropylene amine oxamine (99mTc-HMPAO)-labelled PBMC and [125I]iododeoxyuridine-labelled Con A stimulated lymphoblasts to allogeneic human synovial xenografts in the ears of SCID mice. Successful engraftment of osteoarthritic synovium was achieved in approximately 90% of cases and a connection between the human microvasculature of the xenograft and the circulation of the mouse was shown. Cells were delivered to the xenograft by a system of regional vascular perfusion, thus avoiding the major murine vascular beds. The accumulation of 99mTc-HMPAO-labelled PBMC in mouse ears was monitored in real time. Direct injection of xenograft-bearing ears with recombinant human TNF-alpha, 7 h prior to perfusion, increased the accumulation of both PBMC and lymphoblasts in cytokine-injected ears compared to contralateral control-injected ears. Autoradiography revealed the presence of [125I]iododeoxyuridine-labelled lymphoblasts associated with human microvasculature within the xenograft. However, the increased accumulation of lymphoblasts in cytokine-injected ears occurred in the tissues surrounding the xenograft, where lymphoblasts were associated more often with murine than human vessels. Although the system described offers advantages over similar models, the propensity for mouse endothelium to interact with human leucocytes is likely to be a generic disadvantage for models of human leucocyte recruitment to xenografts in immunodeficient mice.     

Digital image analysis and stereology of angiogenesis in polypoid and nonpolypoid colorectal adenomas

OBJECTIVE: To evaluate 3-dimensional parameters and bidimensional microvascular quantification in the different morphologic presentations of colorectal adenomas. STUDY DESIGN: A study was carried out, including 102 neoplastic colorectal lesions obtained by endoscopy or surgical resection. For the analysis of angiogenesis, immunohistochemistry, digital image analysis, microvascular quantification and stereology were used. RESULTS: Microvascular quantification, volume and microvascular length estimate rose gradually with high grade dysplasia as compared to the low grade ones (P < .001). There was no significant difference in angiogenesis between polypoid and nonpolypoid colorectal adenomas in terms of quantification and microvascular length estimate. CONCLUSION: The use of digital image analysis and stereology added greater objectivity and effectiveness to angiogenic evaluation because they allowed accurate segmentation of hypervascular areas, representation of the characteristic 3-dimensional morphology of the vascular supply and identification of differences in microvascularization in the developmental stages of colorectal cancer. However, no significant relation could be found between macroscopic type and angiogenesis, suggesting that angiogenesis may contribute little to morphogenesis of colorectal adenomas.     

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro–computed tomography (µCT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and µCT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, µCT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7^th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7^th and 10^th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.     

Numerical evaluation reveals the effect of branching morphology on vessel transport properties during angiogenesis

Blood flow governs transport of oxygen and nutrients into tissues. Hypoxic tissues secrete VEGFs to promote angiogenesis during development and in tissue homeostasis. In contrast, tumors enhance pathologic angiogenesis during growth and metastasis, suggesting suppression of tumor angiogenesis could limit tumor growth. In line with these observations, various factors have been identified to control vessel formation in the last decades. However, their impacts on the vascular transport properties of oxygen remain elusive. Here, we take a computational approach to examine the effects of vascular branching on blood flow in the growing vasculature. First of all, we reconstruct a 3D vascular model from the 2D confocal images of the growing vasculature at postnatal day 5 (P5) mouse retina, then simulate blood flow in the vasculatures, which are obtained from the gene targeting mouse models causing hypo- or hyper-branching vascular formation. Interestingly, hyper-branching morphology attenuates effective blood flow at the angiogenic front, likely promoting tissue hypoxia. In contrast, vascular hypo-branching enhances blood supply at the angiogenic front of the growing vasculature. Oxygen supply by newly formed blood vessels improves local hypoxia and decreases VEGF expression at the angiogenic front during angiogenesis. Consistent with the simulation results indicating improved blood flow in the hypo-branching vasculature, VEGF expression around the angiogenic front is reduced in those mouse retinas. Conversely, VEGF expression is enhanced in the angiogenic front of hyper-branching vasculature. Our results indicate the importance of detailed flow analysis in evaluating the vascular transport properties of branching morphology of the blood vessels.     

Estrogen therapy induces collateral and microvascular remodeling

Estrogen increases proliferation and migration of cultured endothelial cells and perfusion of ischemic hindlimbs of rabbits. We tested the hypothesis that estrogen is angiogenic and arteriogenic in the heart during progressive coronary occlusion. Ovariectomized (OVX) and 17beta-estradiol (1 mg.kg(-1).wk(-1) im)-treated OVX (OVX-ES) female New Zealand White rabbits were instrumented with an ameroid occluder on a proximal coronary artery. Four weeks after implantation of an ameroid occluder, we measured myocardial perfusion with microspheres at rest and during adenosine-induced maximal vasodilation. The heart was fixed by perfusion at physiological pressure, and capillary angiogenesis and remodeling were assessed by image analysis of tissue sections in collateral-dependent myocardium. Coronary conductance was higher at rest and during maximal vasodilation in collateral-dependent myocardium of OVX-ES than OVX rabbits. Estrogen treatment increased the wall-to-lumen ratio of collateral vessels while it decreased the wall-to-lumen ratio of noncollateral arteries in normal regions. In normal and collateral-dependent myocardium, mean capillary diameter and capillary volume density were greater in OVX-ES rabbits. However, estrogen had no effect on capillary length density in either region of the myocardium. These data suggest that estrogen induces remodeling of the collateral vasculature and may stimulate growth of the resistance vessels, thereby providing protection during development of a gradual coronary occlusion.     

抗VEGFR-2嵌合Fab抗体对裸鼠肝癌原位移植瘤新生血管生成的影响

目的:检测抗VEGFR-2嵌合Fab抗体对裸鼠肝癌原位移植瘤血管生成的影响.方法:建立裸鼠肝癌H22细胞原位移植瘤模型,随机分成生理盐水组(n=12)和抗体组(n=12).采用免疫组织化学SP染色法对两组肝脏移植瘤进行血管染色,观察其微血管密度(MVD)情况.结果:成功建立裸鼠H22肝癌原位移植瘤模型,HE染色显示肝脏移植瘤为肝细胞肝癌,免疫组化结果显示肝脏实体瘤内微血管密度抗体组较生理盐水组显著性减少(25.64± 1.53 vs 8.65± 1.79,P＜0.05).结论:抗VEGFR-2嵌合Fab抗体能够抑制裸鼠肝癌原位移植瘤的血管生成.     

An antibody for macrophage migration inhibitory factor suppresses tumour growth and inhibits tumour-associated angiogenesis

To verify the role of macrophage migration inhibitory factor (MIF) in tumourigenesis, we examined the effect of an anti-MIF antibody on tumour growth and angiogenesis. We inoculated murine colon adenocarcinoma cell line colon 26 cells subcutaneously into the flank in BALB/c mice. After nine days, we treated tumour-bearing mice with an anti-rat MIF antibody by intraperitoneal injection on days 9, 11, 13, 15, 17, 19 and 21. We found significant inhibition of tumour growth by this treatment from day 15 to day 22. Next, we implanted a chamber filled with colon 26 cells, which only passes soluble factors, in the subcutaneous fascia of the flank, and treated mice with the anti-rat MIF antibody at days 1, 3 and 5. By histological examination at day 6, angiogenesis within the subcutaneous fascia in contact with the chamber was markedly suppressed. In vitro, we added an anti-human MIF antibody to human umbilical vein endothelial cells (HUVEC) to evaluate its effect on cell growth by measurement of [3H]thymidine incorporation. We observed that the anti-MIF antibody significantly suppressed [3H]thymidine uptake by HUVEC. These results suggest the possibility that MIF is involved in tumourigenesis via promotion of angiogenesis.