VEGF‐D deficiency in mice does not affect embryonic or postnatal lymphangiogenesis but reduces lymphatic metastasis

Vascular endothelial growth factor‐D (VEGF‐D) is one of the two ligands of the VEGFR‐3 receptor on lymphatic endothelial cells. Gene‐silencing studies in mice and Xenopus tadpoles recently showed that the role of endogenous VEGF‐D in lymphatic development is moderate. By contrast, exogenous VEGF‐D is capable of stimulating lymphangiogenesis. Nonetheless, its endogenous role in pathological conditions remains largely unknown. Hence, we reassessed its role in disease, using Vegf‐d^null mice. Vegf‐d^null mice were generated that, under physiological conditions, displayed normal embryonic and postnatal lymphangiogenesis and lymphatic remodelling, efficient lymphatic functioning and normal health. Vegf‐d^null mice also reponded normally in models of skin wound healing and healing of infarcted myocardium, despite enhanced expression of VEGF‐D in these models in wild‐type mice. In contrast, Vegf‐d^null mice displayed reduced peritumoral lymphangiogenesis and lymph node metastasis in an orthotopic pancreatic tumour model. Together, our data indicate that endogenous VEGF‐D in mice is dispensible for lymphangiogenesis during development, in postnatal and adult physiology and in several pathological conditions, but significantly contributes to lymphatic metastasis. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

VEGF‐C,VEGF‐D and VEGFR‐3 expression in peripheral neuroblastic tumours

Ramani P, Nash R, Radevsky L, Patel A, Luckett M & Rogers C
(2012) Histopathology
VEGF‐C, VEGF‐D and VEGFR‐3 expression in peripheral neuroblastic tumours Aims: More than 50% of neuroblastomas (NBs) present with haematogenous and/or lymphatic metastasis; however, little is known about the clinicopathological significance in NBs of the key lymphangiogenesis growth factors vascular endothelial growth factor (VEGF)‐C and VEGF‐D and the receptor VEGFR‐3. Methods and results: Ninety‐three NBs and nine ganglioneuromas (GNs) were immunostained for VEGF‐C, VEGF‐D and VEGFR‐3. VEGF‐C and VEGF‐D were present in 76% and 82% of the NBs, respectively. There was no significant difference in VEGF‐C expression between NBs and GNs. VEGF‐D expression was significantly higher in NBs compared with GNs and in MYCN‐amplified NBs. VEGFR‐3 tumoral cell expression (VEGFR‐3c), present in 48% of the NBs, was significantly higher in NBs from children ≥18 months at presentation and those belonging to a high‐risk group. VEGFR‐3 lymphovascular density was increased significantly in NBs compared with GNs and in NBs associated with adverse clinicopathological and biological factors. Lymphovascular invasion, assessed in VEGFR‐3‐stained vessels, was present in ～50% of NBs. Cox regression analyses demonstrated that VEGFR‐3c expression was associated with a significantly shorter event‐free survival and that its effect was independent of the important pathological variable, mitosis–karyorrhexis index. Conclusions: VEGF‐D and VEGFR‐3 up‐regulation support tumour progression in NB and VEGFR‐3c may provide a useful prognostic marker in NBs.     

Accelerated tumour metastasis due to interferon‐γ receptor‐mediated dissociation of perivascular cells from blood vessels

Angiostasis mediated by interferon (IFN)‐γ is a key mechanism of anti‐tumour immunity; however, the effect of IFN‐γ on host vascular endothelial growth factor A (VEGFA)‐expressing cells during tumour progression is still elusive. Here, we developed transgenic mice with IFN‐γ receptor (IFNγR) expression under control of the Vegfa promoter (V‐γR). In these mice, the IFN‐γ responsiveness of VEGFA‐expressing cells led to dramatic growth suppression of transplanted lung carcinoma cells. Surprisingly, increased mortality and tumour metastasis were observed in the tumour‐bearing V‐γR mice, in comparison with the control wild‐type and IFNγR‐deficient mice. Further study showed that perivascular cells were VEGFA‐expressing cells and potential IFN‐γ targets. In vivo, tumour vascular perfusion and pericyte association with blood vessels were massively disrupted in V‐γR mice. In vitro, IFN‐γ inhibited transforming growth factor‐β signalling by upregulating SMAD7, and therefore downregulated N‐cadherin expression in pericytes. Importantly, IFN‐γ neutralization in vivo with a monoclonal antibody reduced tumour metastasis. Together, the results suggest that IFNγR‐mediated dissociation of perivascular cells from blood vessels contributes to the acceleration of tumour metastasis. Thus, the inhibition of tumour growth via IFN‐γ‐induced angiostasis might also accelerate tumour metastasis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Establishment of tooth blood supply and innervation is developmentally regulated and takes place through differential patterning processes

Teeth are richly supported by blood vessels and peripheral nerves. The aim of this study was to describe in detail the developmental time‐course and localization of blood vessels during early tooth formation and to compare that to innervation, as well as to address the putative role of vascular endothelial growth factor (VEGF), which is an essential regulator of vasculature development, in this process. The localization of blood vessels and neurites was compared using double immunofluorescence staining on sections at consecutive stages of the embryonic (E) and postnatal (PN) mandibular first molar tooth germ (E11‐PN7). Cellular mRNA expression domains of VEGF and its signaling receptor VEGFR2 were studied using sectional radioactive in situ hybridization. Expression of VEGF mRNA and the encoded protein were studied by RT‐PCR and western blot analysis, respectively, in the cap and early bell stage tooth germs, respectively. VEGFR2 was immunolocalized on tooth tissue sections. Smooth muscle cells were investigated by anti‐alpha smooth muscle actin (αSMA) antibodies. VEGF showed developmentally regulated epithelial and mesenchymal mRNA expression domains including the enamel knot signaling centers that correlated with the growth and navigation of the blood vessels expressing Vegfr2 and VEGFR2 to the dental papilla and enamel organ. Developing blood vessels were present in the jaw mesenchyme including the presumptive dental mesenchyme before the appearance of the epithelial dental placode and dental neurites. Similarly, formation of a blood vessel plexus around the bud stage tooth germ and ingrowth of vessels into dental papilla at E14 preceded ingrowth of neurites. Subsequently, pioneer blood vessels in the dental papilla started to receive smooth muscle coverage at the early embryonic bell stage. Establishment and patterning of the blood vessels and nerves during tooth formation are developmentally regulated, stepwise processes that likely involve differential patterning mechanisms. Development of tooth vascular supply is proposed to be regulated by local, tooth‐specific regulation by epithelial–mesenchymal tissue interactions and involving tooth target expressed VEGF signaling. Further investigations on tooth vascular development by local VEGF signaling, as well as how tooth innervation and development of blood vessels are integrated with advancing tooth organ formation by local signaling mechanisms, are warranted.     

ANGPTL4–αvβ3 interaction counteracts hypoxia‐induced vascular permeability by modulating Src signalling downstream of vascular endothelial growth factor receptor 2

Dynamic control of endothelial cell junctions is essential for vascular homeostasis and angiogenesis. We recently provided genetic evidence that ANGPTL4 is a key regulator of vascular integrity both during developmental and in hypoxia‐induced pathological conditions. The purpose of the present study was to decipher the molecular mechanisms through which ANGPTL4 regulates vascular integrity. Using surface plasmon resonance and proximity ligation assays, we show that ANGPTL4 binds integrin αvβ3. In vitro and in vivo functional assays with Angptl4‐deficient mice demonstrate that ANGPTL4–αvβ3 interaction is necessary to mediate ANGPTL4 vasoprotective effects. Mechanistically, ANGPTL4–αvβ3 interaction enhances Src recruitment to integrin αvβ3 and inhibits Src signalling downstream of vascular endothelial growth factor receptor 2 (VEFGR2), thereby repressing hypoxia‐induced breakdown of VEGFR2–VE‐cadherin and VEGFR2–αvβ3 complexes. We further demonstrate that intravitreal injection of recombinant human ANGPTL4 limits vascular permeability and leads to increased adherens junction and tight junction integrity. These findings identify a novel mechanism by which ANGPTL4 counteracts hypoxia‐driven vascular permeability through integrin αvβ3 binding, modulation of VEGFR2–Src kinase signalling, and endothelial junction stabilization. We further demonstrate that Angptl4‐deficient mice show increased vascular leakage in vivo in a model of laser‐induced choroidal neovascularization, indicating that this newly identified ANGPTL4–αvβ3 axis might be a target for pharmaceutical intervention in pathological conditions. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Transgenic overexpression of granulocyte macrophage-colony stimulating factor in the lung prevents hyperoxic lung injury

Granulocyte macrophage-colony stimulating factor (GM-CSF) plays an important role in pulmonary homeostasis, with effects on both alveolar macrophages and alveolar epithelial cells. We hypothesized that overexpression of GM-CSF in the lung would protect mice from hyperoxic lung injury by limiting alveolar epithelial cell injury. Wild-type C57BL/6 mice and mutant mice in which GM-CSF was overexpressed in the lung under control of the SP-C promoter (SP-C-GM mice) were placed in >95% oxygen. Within 6 days, 100% of the wild-type mice had died, while 70% of the SP-C-GM mice remained alive after 10 days in hyperoxia. Histological assessment of the lungs at day 4 revealed less disruption of the alveolar wall in SP-C-GM mice compared to wild-type mice. The concentration of albumin in bronchoalveolar lavage fluid after 4 days in hyperoxia was significantly lower in SP-C-GM mice than in wild-type mice, indicating preservation of alveolar epithelial barrier properties in the SP-C-GM mice. Alveolar fluid clearance was preserved in SP-C-GM mice in hyperoxia, but decreased significantly in hyperoxia-exposed wild-type mice. Staining of lung tissue for caspase 3 demonstrated increased apoptosis in alveolar wall cells in wild-type mice in hyperoxia compared to mice in room air. In contrast, SP-C-GM mice exposed to hyperoxia demonstrated only modest increase in alveolar wall apoptosis compared to room air. Systemic treatment with GM-CSF (9 micro g/kg/day) during 4 days of hyperoxic exposure resulted in decreased apoptosis in the lungs compared to placebo. In studies using isolated murine type II alveolar epithelial cells, treatment with GM-CSF greatly reduced apoptosis in response to suspension culture. In conclusion, overexpression of GM-CSF enhances survival of mice in hyperoxia; this effect may be explained by preservation of alveolar epithelial barrier function and fluid clearance, at least in part because of reduction in hyperoxia-induced apoptosis of cells in the alveolar wall.     

Deficiency of bone marrow β3‐integrin enhances non‐functional neovascularization

β3‐Integrin is a cell surface adhesion and signalling molecule important in the regulation of tumour angiogenesis. Mice with a global deficiency in β3‐integrin show increased pathological angiogenesis, most likely due to increased vascular endothelial growth factor receptor 2 expression on β3‐null endothelial cells. Here we transplanted β3‐null bone marrow (BM) into wild‐type (WT) mice to dissect the role of BM β3‐integrin deficiency in pathological angiogenesis. Mice transplanted with β3‐null bone marrow show significantly enhanced angiogenesis in subcutaneous B16F0 melanoma and Lewis lung carcinoma (LLC) cell models and in B16F0 melanoma lung metastasis when compared with tumours grown in mice transplanted with WT bone marrow. The effect of bone marrow β3‐integrin deficiency was also assessed in the RIPTAg mouse model of pancreatic tumour growth. Again, angiogenesis in mice lacking BM β3‐integrin was enhanced. However, tumour weight between the groups was not significantly altered, suggesting that the enhanced blood vessel density in the mice transplanted with β3‐null bone marrow was not functional. Indeed, we demonstrate that in mice transplanted with β3‐null bone marrow a significant proportion of tumour blood vessels are non‐functional when compared with tumour blood vessels in WT‐transplanted controls. Furthermore, β3‐null‐transplanted mice showed an increased angiogenic response to VEGF in vivo when compared with WT‐transplanted animals. BM β3‐integrin deficiency affects the mobilization of progenitor cells to the peripheral circulation. We show that VEGF‐induced mobilization of endothelial progenitor cells is enhanced in mice transplanted with β3‐null bone marrow when compared with WT‐transplanted controls, suggesting a possible mechanism underlying the increased blood vessel density seen in β3‐null‐transplanted mice. In conclusion, although BM β3‐integrin is not required for pathological angiogenesis, our studies demonstrate a role for BM β3‐integrin in VEGF‐induced mobilization of bone marrow‐derived cells to the peripheral circulation and for the functionality of those vessels in which BM‐derived cells become incorporated. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

NLRP3 inflammasome mediates interleukin‐1β production in immune cells in response to Acinetobacter baumannii and contributes to pulmonary inflammation in mice

Acinetobacter baumannii is a multi‐drug resistant, Gram‐negative bacteria and infection with this organism is one of the major causes of mortality in intensive care units. Inflammasomes are multiprotein oligomers that include caspase‐1, and their activation is required for maturation of interleukin‐1β (IL‐1β). Inflammasome signalling is involved in host defences against various microbial infections, but the precise mechanism by which A. baumannii activates inflammasomes and the roles of relevant signals in host defence against pulmonary A. baumannii infection are unknown. Our results showed that NLRP3, ASC and caspase‐1, but not NLRC4, are required for A. baumannii‐induced production of IL‐1β in macrophages. An inhibitor assay revealed that various pathways, including P2X7R, K⁺ efflux, reactive oxygen species production and release of cathepsins, are involved in IL‐1β production in macrophages in response to A. baumannii. Interleukin‐1β production in bronchoalveolar lavage (BAL) fluid was impaired in NLRP3‐deficient and caspase‐1/11‐deficient mice infected with A. baumannii, compared with that in wild‐type (WT) mice. However, the bacterial loads in BAL fluid and lungs were comparable between WT and NLRP3‐deficient or caspase‐1/11‐deficient mice. The severity of lung pathology was reduced in NLRP3‐ deficient, caspase‐1/11‐ deficient and IL‐1‐receptor‐deficient mice, although the recruitment of immune cells and production of inflammatory cytokines and chemokines were not altered in these mice. These findings indicate that A. baumannii leads to the activation of NLRP3 inflammasome, which mediates IL‐1β production and lung pathology.     

Apoptosis in neonatal murine lung exposed to hyperoxia

Exposure to high concentrations of oxygen in the neonatal period may impair lung growth and is a major contributing factor to the development of bronchopulmonary dysplasia. Cell death from hyperoxic injury may occur through either an apoptotic or nonapoptotic pathway, and we were interested in determining the type of cell death that occurs in the lung of neonatal mice exposed to hyperoxia. We found increased levels of Bax messenger RNA, a gene associated with apoptosis, in the lungs of neonatal mice born and raised in 92% hyperoxia. We next determined the extent of apoptosis taking place in the lungs of neonatal mice exposed to hyperoxia using terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling in 3.5-, 4.5-, and 5.5-d-old neonatal lung. The number of apoptotic cells in peripheral lung was significantly higher in the 3.5-, 4.5-, and 5.5-d-old mice treated with oxygen compared with that in the room-air control mice. Further, the number of apoptotic cells in the lung increased with longer exposure duration. In murine lung bronchus cells exposed to hyperoxia, growth arrest occurred after 48 h of oxygen exposure. Using annexin V binding, necrotic cell death was found to be the major form of cell death in these cells after 72 h of hyperoxic exposure. We conclude that 92% hyperoxia causes significant lung injury in neonatal mice exposed to hyperoxia, and that the number of apoptotic cells in the lung increases the longer the duration of exposure. The increase in apoptosis from hyperoxic exposure during a critical period of lung development may be an important factor in the impaired lung growth and remodeling that occur in animals exposed to high oxygen concentrations. Finally, it appears that hyperoxic injured cells in neonatal lung undergo both apoptotic and nonapoptotic cell death.     

Complementary actions of VEGF and angiopoietin-1 on blood vessel growth and leakage

Vascular endothelial growth factor (VEGF) and Angiopoietins are families of vascular-specific growth factors that regulate blood vessel growth, maturation and function. To learn more about the effects of these factors in vivo, we have overexpressed VEGF-A or Angiopoietin-1 (Ang1) in two systems in mice, and examined the effects on blood vessel growth and function. In one set of studies, VEGF, Ang1, or both factors, were transgenically overexpressed in the skin under the keratin-14 (K14) promoter. The skin of mice overexpressing VEGF (K14-VEGF) had numerous tortuous, capillary-sized vessels which were leaky to the plasma tracer Evans blue under baseline conditions. In contrast, the skin of mice overexpressing Ang1 (K14-Ang1) had enlarged dermal vessels without a significant increase in vessel number. These enlarged vessels were less leaky than those of wild-type mice in response to inflammatory stimuli. In double transgenic mice overexpressing VEGF and Ang1, the size and number of skin vessels were both increased; however, the vessels were not leaky. In a second set of studies, VEGF or Ang1 was systemically delivered using an adenoviral approach. Intravenous injection of adenovirus encoding VEGF (Adeno-VEGF) resulted in widespread tissue oedema within 1-2 days after administration, whereas injection of Adeno-Ang1 resulted in the skin vessels becoming less leaky in response to topical inflammatory stimuli or local injection of VEGF. The decreased leakage was not accompanied by morphological changes. Thus, overexpressing VEGF appears to promote growth of new vessels accompanied by plasma leakage, whereas overexpressing Ang1 promotes the enlargement of existing vessels and a resistance to leakage. Further understanding of the interrelationship of these factors during normal development could lead to their application in the treatment of ischaemic diseases.     

Increased hyperoxia-induced lung injury in nitric oxide synthase 2 null mice is mediated via angiopoietin 2

Supplemental oxygen is frequently prescribed. However, prolonged exposure to high concentrations of oxygen causes hyperoxic acute lung injury (HALI), which manifests as acute respiratory distress syndrome in adults and leads to bronchopulmonary dysplasia in newborns (NBs). Nitric oxide (NO), NO synthases (NOSs), and angiopoietin (Ang) 2 have been implicated in the pathogenesis of HALI. However, the mechanisms of the contributions of NOS/NO and the relationship(s) between NOS/NO and Ang2 have not been addressed. In addition, the relevance of these moieties in adults and NBs has not been evaluated. To address these issues, we compared the responses in hyperoxia of wild-type (NOS [+/+]) and NOS null (-/-) young adult and NB mice. When compared with NOS2(+/+) adult controls, NOS2(-/-) animals manifest exaggerated alveolar-capillary protein leak and premature death. These responses were associated with enhanced levels of structural cell death, enhanced expression of proapoptotic regulatory proteins, and Ang2. Importantly, silencing RNA knockdown of Ang2 decreased the levels of cell death and the expression of proapoptotic mediators. These effects were at least partially NOS2 specific, and were development dependent, because survival was similar in adult NOS3(+/+) and NOS3(-/-) mice and NB NOS2(+/+) and NOS2(-/-) mice, respectively. These studies demonstrate that NOS2 plays an important protective role in HALI in adult animals. They also demonstrate that this response is mediated, at least in part, by the ability of NOS2 to inhibit hyperoxia-induced Ang2 production and thereby decrease Ang2-induced tissue injury.     

Inhibition of transforming growth factor-�� signalling attenuates interleukin (IL)-18 plus IL-2-induced interstitial lung disease in mice

Interstitial lung disease (ILD) is an intractable disease induced by various factors in humans. However, there is no universally effective treatment for ILD. In this study, we investigated the role of transforming growth factor (TGF)‐β signalling in the pathogenesis of ILD by using model mice. Injection of interleukin (IL)‐18 plus IL‐2 in C57BL6 (B6) mice resulted in acute ILD by infiltration of natural killer (NK) cells and a significant increase of TGF‐β mRNA in the lung. To examine the pathogenetic role of TGF‐β in ILD mice, we used SB‐431542 (4‐[4‐(1,3‐benzodioxol‐5‐yl)‐5‐(2‐pyridinyl)‐1H‐imidazol‐2‐yl]‐benzamide), which is a potent and selective inhibitor of TGF‐β receptor I (TβRI), also known as activin receptor‐like kinase 5 (ALK5). Treatment of B6‐ILD mice with SB‐431542 resulted in improvement of ILD, delay in mortality, reduction of the expression of interferon (IFN)‐γ and IL‐6 in the lungs. The same treatment also decreased significantly the percentage of natural killer (NK) cells in the lungs (P < 0·05) and mRNA expression levels of certain chemokines such as CCL2, CCL3, CCL4, CCL5 and CXCL10 in B6‐ILD. These findings were confirmed by IL‐18 plus IL‐2 treatment of Smad3‐deficient (Smad3^–/–) mice (P < 0·05). Our results showed that inhibition of TGF‐β signalling reduced the percentage of NK cells and the expression of certain chemokines in the lungs, resulting in improvement of ILD. The findings suggest that TGF‐β signalling may play an important role in the pathogenesis of IL‐18 plus IL‐2‐induced ILD in mice.     

Targeting galectin‐1‐induced angiogenesis mitigates the severity of endometriosis

Endometriosis is characterized by the presence of endometrial tissue outside the uterus that causes severe pelvic pain and infertility in women of reproductive age. Although not completely understood, the pathophysiology of the disease involves chronic dysregulation of inflammatory and vascular signalling. In the quest for novel therapeutic targets, we investigated the involvement of galectin‐1 (Gal‐1), an endogenous glycan‐binding protein endowed with both immunosuppressive and pro‐angiogenic activities, in the pathophysiology of endometriotic lesions. Here we show that Gal‐1 is selectively expressed in stromal and endothelial cells of human endometriotic lesions. Using an experimental endometriosis model induced in wild‐type and Gal‐1‐deficient (Lgals1^?/?) mice, we showed that this lectin orchestrates the formation of vascular networks in endometriotic lesions in vivo, facilitating their ectopic growth independently of vascular endothelial growth factor (VEGF) and the keratinocyte‐derived CXC‐motif (CXC‐KC) chemokine. Targeting Gal‐1 using a specific neutralizing mAb reduced the size and vascularized area of endometriotic lesions within the peritoneal compartment. These results underline the essential role of Gal‐1 during endometriosis and validate this lectin as a possible target for the treatment of disease. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

NF‐κB‐inducing kinase is a key regulator of inflammation‐induced and tumour‐associated angiogenesis

Angiogenesis is essential during development and in pathological conditions such as chronic inflammation and cancer progression. Inhibition of angiogenesis by targeting vascular endothelial growth factor (VEGF) blocks disease progression, but most patients eventually develop resistance which may result from compensatory signalling pathways. In endothelial cells (ECs), expression of the pro‐angiogenic chemokine CXCL12 is regulated by non‐canonical nuclear factor (NF)‐κB signalling. Here, we report that NF‐κB‐inducing kinase (NIK) and subsequent non‐canonical NF‐κB signalling regulate both inflammation‐induced and tumour‐associated angiogenesis. NIK is highly expressed in endothelial cells (ECs) in tumour tissues and inflamed rheumatoid arthritis synovial tissue. Furthermore, non‐canonical NF‐κB signalling in human microvascular ECs significantly enhanced vascular tube formation, which was completely blocked by siRNA targeting NIK. Interestingly, Nik^?/? mice exhibited normal angiogenesis during development and unaltered TNFα‐ or VEGF‐induced angiogenic responses, whereas angiogenesis induced by non‐canonical NF‐κB stimuli was significantly reduced. In addition, angiogenesis in experimental arthritis and a murine tumour model was severely impaired in these mice. These studies provide evidence for a role of non‐canonical NF‐κB signalling in pathological angiogenesis, and identify NIK as a potential therapeutic target in chronic inflammatory diseases and tumour neoangiogenesis. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Investigating intracranial tumour growth patterns with multiparametric MRI incorporating Gd‐DTPA and USPIO‐enhanced imaging

High grade and metastatic brain tumours exhibit considerable spatial variations in proliferation, angiogenesis, invasion, necrosis and oedema. Vascular heterogeneity arising from vascular co‐option in regions of invasive growth (in which the blood–brain barrier remains intact) and neoangiogenesis is a major challenge faced in the assessment of brain tumours by conventional MRI. A multiparametric MRI approach, incorporating native measurements and both Gd‐DTPA (Magnevist) and ultrasmall superparamagnetic iron oxide (P904)‐enhanced imaging, was used in combination with histogram and unsupervised cluster analysis using a k‐means algorithm to examine the spatial distribution of vascular parameters, water diffusion characteristics and invasion in intracranially propagated rat RG2 gliomas and human MDA‐MB‐231 LM2–4 breast adenocarcinomas in mice. Both tumour models presented with higher ΔR₁ (the change in transverse relaxation rate R₁ induced by Gd‐DTPA), fractional blood volume (fBV) and apparent diffusion coefficient than uninvolved regions of the brain. MDA‐MB‐231 LM2–4 tumours were less densely cellular than RG2 tumours and exhibited substantial local invasion, associated with oedema, whereas invasion in RG2 tumours was minimal. These additional features were reflected in the more heterogeneous appearance of MDA‐MB‐231 LM2–4 tumours on T₂‐weighted images and maps of functional MRI parameters. Unsupervised cluster analysis separated subregions with distinct functional properties; areas with a low fBV and relatively impermeable blood vessels (low ΔR₁) were predominantly located at the tumour margins, regions of MDA‐MB‐231 LM2–4 tumours with relatively high levels of water diffusion and low vascular permeability and/or fBV corresponded to histologically identified regions of invasion and oedema, and areas of mismatch between vascular permeability and blood volume were identified. We demonstrate that dual contrast MRI and evaluation of tissue diffusion properties, coupled with cluster analysis, allows for the assessment of heterogeneity within invasive brain tumours and the designation of functionally diverse subregions that may provide more informative predictive biomarkers.