Identification of human brain tumour initiating cells

The cancer stem cell (CSC) hypothesis suggests that neoplastic clones are maintained exclusively by a rare fraction of cells with stem cell properties. Although the existence of CSCs in human leukaemia is established, little evidence exists for CSCs in solid tumours, except for breast cancer. Recently, we prospectively isolated a CD133+ cell subpopulation from human brain tumours that exhibited stem cell properties in vitro. However, the true measures of CSCs are their capacity for self renewal and exact recapitulation of the original tumour. Here we report the development of a xenograft assay that identified human brain tumour initiating cells that initiate tumours in vivo. Only the CD133+ brain tumour fraction contains cells that are capable of tumour initiation in NOD-SCID (non-obese diabetic, severe combined immunodeficient) mouse brains. Injection of as few as 100 CD133+ cells produced a tumour that could be serially transplanted and was a phenocopy of the patient's original tumour, whereas injection of 10(5) CD133- cells engrafted but did not cause a tumour. Thus, the identification of brain tumour initiating cells provides insights into human brain tumour pathogenesis, giving strong support for the CSC hypothesis as the basis for many solid tumours, and establishes a previously unidentified cellular target for more effective cancer therapies.     

A human colon cancer cell capable of initiating tumour growth in immunodeficient mice

Colon cancer is one of the best-understood neoplasms from a genetic perspective, yet it remains the second most common cause of cancer-related death, indicating that some of its cancer cells are not eradicated by current therapies. What has yet to be established is whether every colon cancer cell possesses the potential to initiate and sustain tumour growth, or whether the tumour is hierarchically organized so that only a subset of cells--cancer stem cells--possess such potential. Here we use renal capsule transplantation in immunodeficient NOD/SCID mice to identify a human colon cancer-initiating cell (CC-IC). Purification experiments established that all CC-ICs were CD133+; the CD133- cells that comprised the majority of the tumour were unable to initiate tumour growth. We calculated by limiting dilution analysis that there was one CC-IC in 5.7 x 10(4) unfractionated tumour cells, whereas there was one CC-IC in 262 CD133+ cells, representing >200-fold enrichment. CC-ICs within the CD133+ population were able to maintain themselves as well as differentiate and re-establish tumour heterogeneity upon serial transplantation. The identification of colon cancer stem cells that are distinct from the bulk tumour cells provides strong support for the hierarchical organization of human colon cancer, and their existence suggests that for therapeutic strategies to be effective, they must target the cancer stem cells.     

Identification of cells initiating human melanomas

Tumour-initiating cells capable of self-renewal and differentiation, which are responsible for tumour growth, have been identified in human haematological malignancies and solid cancers. If such minority populations are associated with tumour progression in human patients, specific targeting of tumour-initiating cells could be a strategy to eradicate cancers currently resistant to systemic therapy. Here we identify a subpopulation enriched for human malignant-melanoma-initiating cells (MMIC) defined by expression of the chemoresistance mediator ABCB5 (refs 7, 8) and show that specific targeting of this tumorigenic minority population inhibits tumour growth. ABCB5+ tumour cells detected in human melanoma patients show a primitive molecular phenotype and correlate with clinical melanoma progression. In serial human-to-mouse xenotransplantation experiments, ABCB5+ melanoma cells possess greater tumorigenic capacity than ABCB5- bulk populations and re-establish clinical tumour heterogeneity. In vivo genetic lineage tracking demonstrates a specific capacity of ABCB5+ subpopulations for self-renewal and differentiation, because ABCB5+ cancer cells generate both ABCB5+ and ABCB5- progeny, whereas ABCB5- tumour populations give rise, at lower rates, exclusively to ABCB5- cells. In an initial proof-of-principle analysis, designed to test the hypothesis that MMIC are also required for growth of established tumours, systemic administration of a monoclonal antibody directed at ABCB5, shown to be capable of inducing antibody-dependent cell-mediated cytotoxicity in ABCB5+ MMIC, exerted tumour-inhibitory effects. Identification of tumour-initiating cells with enhanced abundance in more advanced disease but susceptibility to specific targeting through a defining chemoresistance determinant has important implications for cancer therapy.     

Glioma stem cells promote radioresistance by preferential activation of the DNA damage response

Ionizing radiation represents the most effective therapy for glioblastoma (World Health Organization grade IV glioma), one of the most lethal human malignancies, but radiotherapy remains only palliative because of radioresistance. The mechanisms underlying tumour radioresistance have remained elusive. Here we show that cancer stem cells contribute to glioma radioresistance through preferential activation of the DNA damage checkpoint response and an increase in DNA repair capacity. The fraction of tumour cells expressing CD133 (Prominin-1), a marker for both neural stem cells and brain cancer stem cells, is enriched after radiation in gliomas. In both cell culture and the brains of immunocompromised mice, CD133-expressing glioma cells survive ionizing radiation in increased proportions relative to most tumour cells, which lack CD133. CD133-expressing tumour cells isolated from both human glioma xenografts and primary patient glioblastoma specimens preferentially activate the DNA damage checkpoint in response to radiation, and repair radiation-induced DNA damage more effectively than CD133-negative tumour cells. In addition, the radioresistance of CD133-positive glioma stem cells can be reversed with a specific inhibitor of the Chk1 and Chk2 checkpoint kinases. Our results suggest that CD133-positive tumour cells represent the cellular population that confers glioma radioresistance and could be the source of tumour recurrence after radiation. Targeting DNA damage checkpoint response in cancer stem cells may overcome this radioresistance and provide a therapeutic model for malignant brain cancers.     

APC mutations occur early during colorectal tumorigenesis.

Human tumorigenesis is associated with the accumulation of mutations both in oncogenes and in tumour suppressor genes. But in no common adult cancer have the mutations that are critical in the early stages of the tumorigenic process been defined. We have attempted to determine if mutations of the APC gene play such a role in human colorectal tumours, which evolve from small benign tumours (adenomas) to larger malignant tumours (carcinomas) over the course of several decades. Here we report that sequence analysis of 41 colorectal tumours revealed that the majority of colorectal carcinomas (60%) and adenomas (63%) contained a mutated APC gene. Furthermore, the APC gene met two criteria of importance for tumour initiation. First, mutations of this gene were found in the earliest tumours that could be analysed, including adenomas as small as 0.5 cm in diameter. Second, the frequency of such mutations remained constant as tumours progressed from benign to malignant stages. These data provide strong evidence that mutations of the APC gene play a major role in the early development of colorectal neoplasms.     

Colorectal carcinomas in mice lacking the catalytic subunit of PI(3)Kgamma

Phosphoinositide-3-OH kinases (PI(3)Ks) constitute a family of evolutionarily conserved lipid kinases that regulate a vast array of fundamental cellular responses, including proliferation, transformation, differentiation and protection from apoptosis. PI(3)K-mediated activation of the cell survival kinase PKB/Akt, and negative regulation of PI(3)K signalling by the tumour suppressor PTEN (refs 3, 4) are key regulatory events in tumorigenesis. Thus, a model has arisen that PI(3)Ks promote development of cancers. Here we report that genetic inactivation of the p110gamma catalytic subunit of PI(3)Kgamma (ref. 8) leads to development of invasive colorectal adenocarcinomas in mice. In humans, p110gamma protein expression is lost in primary colorectal adenocarcinomas from patients and in colon cancer cell lines. Overexpression of wild-type or kinase-dead p110gamma in human colon cancer cells with mutations of the tumour suppressors APC and p53, or the oncogenes beta-catenin and Ki-ras, suppressed tumorigenesis. Thus, loss of p110gamma in mice leads to spontaneous, malignant epithelial tumours in the colorectum and p110gamma can block the growth of human colon cancer cells.     

Cutaneous cancer stem cell maintenance is dependent on beta-catenin signalling

Continuous turnover of epithelia is ensured by the extensive self-renewal capacity of tissue-specific stem cells. Similarly, epithelial tumour maintenance relies on cancer stem cells (CSCs), which co-opt stem cell properties. For most tumours, the cellular origin of these CSCs and regulatory pathways essential for sustaining stemness have not been identified. In murine skin, follicular morphogenesis is driven by bulge stem cells that specifically express CD34. Here we identify a population of cells in early epidermal tumours characterized by phenotypic and functional similarities to normal bulge skin stem cells. This population contains CSCs, which are the only cells with tumour initiation properties. Transplants derived from these CSCs preserve the hierarchical organization of the primary tumour. We describe beta-catenin signalling as being essential in sustaining the CSC phenotype. Ablation of the beta-catenin gene results in the loss of CSCs and complete tumour regression. In addition, we provide evidence for the involvement of increased beta-catenin signalling in malignant human squamous cell carcinomas. Because Wnt/beta-catenin signalling is not essential for normal epidermal homeostasis, such a mechanistic difference may thus be targeted to eliminate CSCs and consequently eradicate squamous cell carcinomas.     

Localization of the gene for familial adenomatous polyposis on chromosome 5

Colorectal cancer is the second most common cancer in the United Kingdom and other developed countries in the West. Although it is usually not familial, there is a rare dominantly inherited susceptibility to colon cancer, familial adenomatous polyposis (FAP; also often previously called familial polyposis coli). During adolescence affected individuals develop from a few hundred to over a thousand adenomatous polyps in their large bowel. These are sufficiently likely to give rise to adenocarcinomas to make prophylactic removal of the colon usual in diagnosed FAP individuals. Adenomas may occur elsewhere in the gastrointestinal tract and the condition is often associated with other extracolonic lesions, such as epidermoid cysts, jaw osteomata and fibrous desmoid tumours. Adenomata have been suggested to be precancerous states for most colorectal tumours. Knudson has suggested that the mutation for a dominantly inherited cancer susceptibility may be the first step in a recessive change in the tumour cells, and that the same gene may be involved in both familial and non-familial cases of a given tumour. Following up a case report of an interstitial deletion of chromosome 5 in a mentally retarded individual with multiple developmental abnormalities and FAP, we have now shown that the FAP gene is on chromosome 5, most probably near bands 5q21-q22.     

Glioblastoma stem-like cells give rise to tumour endothelium

Glioblastoma (GBM) is among the most aggressive of human cancers. A key feature of GBMs is the extensive network of abnormal vasculature characterized by glomeruloid structures and endothelial hyperplasia. Yet the mechanisms of angiogenesis and the origin of tumour endothelial cells remain poorly defined. Here we demonstrate that a subpopulation of endothelial cells within glioblastomas harbour the same somatic mutations identified within tumour cells, such as amplification of EGFR and chromosome 7. We additionally demonstrate that the stem-cell-like CD133(+) fraction includes a subset of vascular endothelial-cadherin (CD144)-expressing cells that show characteristics of endothelial progenitors capable of maturation into endothelial cells. Extensive in vitro and in vivo lineage analyses, including single cell clonal studies, further show that a subpopulation of the CD133(+) stem-like cell fraction is multipotent and capable of differentiation along tumour and endothelial lineages, possibly via an intermediate CD133(+)/CD144(+) progenitor cell. The findings are supported by genetic studies of specific exons selected from The Cancer Genome Atlas, quantitative FISH and comparative genomic hybridization data that demonstrate identical genomic profiles in the CD133(+) tumour cells, their endothelial progenitor derivatives and mature endothelium. Exposure to the clinical anti-angiogenesis agent bevacizumab or to a γ-secretase inhibitor as well as knockdown shRNA studies demonstrate that blocking VEGF or silencing VEGFR2 inhibits the maturation of tumour endothelial progenitors into endothelium but not the differentiation of CD133(+) cells into endothelial progenitors, whereas γ-secretase inhibition or NOTCH1 silencing blocks the transition into endothelial progenitors. These data may provide new perspectives on the mechanisms of failure of anti-angiogenesis inhibitors currently in use. The lineage plasticity and capacity to generate tumour vasculature of the putative cancer stem cells within glioblastoma are novel findings that provide new insight into the biology of gliomas and the definition of cancer stemness, as well as the mechanisms of tumour neo-angiogenesis.     

Bv8 regulates myeloid-cell-dependent tumour angiogenesis

Bone-marrow-derived cells facilitate tumour angiogenesis, but the molecular mechanisms of this facilitation are incompletely understood. We have previously shown that the related EG-VEGF and Bv8 proteins, also known as prokineticin 1 (Prok1) and prokineticin 2 (Prok2), promote both tissue-specific angiogenesis and haematopoietic cell mobilization. Unlike EG-VEGF, Bv8 is expressed in the bone marrow. Here we show that implantation of tumour cells in mice resulted in upregulation of Bv8 in CD11b+Gr1+ myeloid cells. We identified granulocyte colony-stimulating factor as a major positive regulator of Bv8 expression. Anti-Bv8 antibodies reduced CD11b+Gr1+ cell mobilization elicited by granulocyte colony-stimulating factor. Adenoviral delivery of Bv8 into tumours was shown to promote angiogenesis. Anti-Bv8 antibodies inhibited growth of several tumours in mice and suppressed angiogenesis. Anti-Bv8 treatment also reduced CD11b+Gr1+ cells, both in peripheral blood and in tumours. The effects of anti-Bv8 antibodies were additive to those of anti-Vegf antibodies or cytotoxic chemotherapy. Thus, Bv8 modulates mobilization of CD11b+Gr1+ cells from the bone marrow during tumour development and also promotes angiogenesis locally.     

Characterization of a common precursor population for dendritic cells

Dendritic cells (DCs) are essential for the establishment of immune responses against pathogens and tumour cells, and thus have great potential as tools for vaccination and cancer immunotherapy trials. Experimental evidence has led to a dual DC differentiation model, which involves the existence of both myeloid- and lymphoid-derived DCs. But this concept has been challenged by recent reports demonstrating that both CD8- and CD8+ DCs, considered in mice as archetypes of myeloid and lymphoid DCs respectively, can be generated from either lymphoid or myeloid progenitors. The issue of DC physiological derivation therefore remains an open question. Here we report the characterization of a DC-committed precursor population, which has the capacity to generate all the DC subpopulations present in mouse lymphoid organs---including CD8- and CD8+ DCs, as well as the B220+ DC subset---but which is devoid of myeloid or lymphoid differentiation potential. These data support an alternative model of DC development, in which there is an independent, common DC differentiation pathway.     

Chromosome 5 allele loss in human colorectal carcinomas

That the sporadic and inherited forms of a particular cancer could both result from mutations in the same gene was first proposed by Knudson. He further proposed that these mutations act recessively at the cellular level, and that both copies of the gene must be lost for the cancer to develop. In sporadic cases both events occur somatically whereas in dominant familial cases susceptibility is inherited through a germline mutation and the cancer develops after a somatic change in the homologous allele. This model has since been substantiated in the case of retinoblastoma, Wilms tumour, acoustic neuroma and several other tumours, in which loss of heterozygosity was shown in tumour material compared to normal tissue from the same patient. The dominantly inherited disorder, familial adenomatous polyposis (FAP, also called familial polyposis coli), which gives rise to multiple adenomatous polyps in the colon that have a relatively high probability of progressing to a malignant adenocarcinoma, provides a basis for studying recessive genes in the far more common colorectal carcinomas using this approach. Following a clue as to the location of the FAP gene given by a case report of an individual with an interstitial deletion of chromosome 5q, who had FAP and multiple developmental abnormalities, we have examined sporadic colorectal adenocarcinomas for loss of alleles on chromosome 5. Using a highly polymorphic 'minisatellite' probe which maps to chromosome 5q we have shown that at least 20% of this highly heterogeneous set of tumours lose one of the alleles present in matched normal tissue. This parallels the assignment of the FAP gene to chromosome 5 (see accompanying paper) and suggests that becoming recessive for this gene may be a critical step in the progression of a relatively high proportion of colorectal cancers.     

Disulphide-isomerase-enabled shedding of tumour-associated NKG2D ligands

Tumour-associated ligands of the activating NKG2D (natural killer group 2, member D; also called KLRK1) receptor-which are induced by genotoxic or cellular stress-trigger activation of natural killer cells and co-stimulation of effector T cells, and may thus promote resistance to cancer. However, many progressing tumours in humans counter this anti-tumour activity by shedding the soluble major histocompatibility complex class-I-related ligand MICA, which induces internalization and degradation of NKG2D and stimulates population expansions of normally rare NKG2D+CD4+ T cells with negative regulatory functions. Here we show that on the surface of tumour cells, MICA associates with endoplasmic reticulum protein 5 (ERp5; also called PDIA6 or P5), which, similar to protein disulphide isomerase, usually assists in the folding of nascent proteins inside cells. Pharmacological inhibition of thioreductase activity and ERp5 gene silencing revealed that cell-surface ERp5 function is required for MICA shedding. ERp5 and membrane-anchored MICA form transitory mixed disulphide complexes from which soluble MICA is released after proteolytic cleavage near the cell membrane. Reduction of the seemingly inaccessible disulphide bond in the membrane-proximal alpha3 domain of MICA must involve a large conformational change that enables proteolytic cleavage. These results uncover a molecular mechanism whereby domain-specific deconstruction regulates MICA protein shedding, thereby promoting tumour immune evasion, and identify surface ERp5 as a strategic target for therapeutic intervention.     

Deleted in colorectal carcinoma suppresses metastasis in p53-deficient mammary tumours

Since its discovery in the early 1990s the deleted in colorectal cancer (DCC) gene, located on chromosome 18q21, has been proposed as a tumour suppressor gene as its loss is implicated in the majority of advanced colorectal and many other cancers. DCC belongs to the family of netrin 1 receptors, which function as dependence receptors as they control survival or apoptosis depending on ligand binding. However, the role of DCC as a tumour suppressor remains controversial because of the rarity of DCC-specific mutations and the presence of other tumour suppressor genes in the same chromosomal region. Here we show that in a mouse model of mammary carcinoma based on somatic inactivation of p53, additional loss of DCC promotes metastasis formation without affecting the primary tumour phenotype. Furthermore, we demonstrate that in cell cultures derived from p53-deficient mouse mammary tumours DCC expression controls netrin-1-dependent cell survival, providing a mechanistic basis for the enhanced metastatic capacity of tumour cells lacking DCC. Consistent with this idea, in vivo tumour-cell survival is enhanced by DCC loss. Together, our data support the function of DCC as a context-dependent tumour suppressor that limits survival of disseminated tumour cells.     

IL-23 promotes tumour incidence and growth

Chronic inflammation has long been associated with increased incidence of malignancy and similarities in the regulatory mechanisms have been suggested for more than a century. Infiltration of innate immune cells, elevated activities of matrix metalloproteases and increased angiogenesis and vasculature density are a few examples of the similarities between chronic and tumour-associated inflammation. Conversely, the elimination of early malignant lesions by immune surveillance, which relies on the cytotoxic activity of tumour-infiltrating T cells or intra-epithelial lymphocytes, is thought to be rate-limiting for the risk to develop cancer. Here we show a molecular connection between the rise in tumour-associated inflammation and a lack of tumour immune surveillance. Expression of the heterodimeric cytokine interleukin (IL)-23, but not of its close relative IL-12, is increased in human tumours. Expression of these cytokines antagonistically regulates local inflammatory responses in the tumour microenvironment and infiltration of intra-epithelial lymphocytes. Whereas IL-12 promotes infiltration of cytotoxic T cells, IL-23 promotes inflammatory responses such as upregulation of the matrix metalloprotease MMP9, and increases angiogenesis but reduces CD8 T-cell infiltration. Genetic deletion or antibody-mediated elimination of IL-23 leads to increased infiltration of cytotoxic T cells into the transformed tissue, rendering a protective effect against chemically induced carcinogenesis. Finally, transplanted tumours are growth-restricted in hosts depleted for IL-23 or in IL-23-receptor-deficient mice. Although many strategies for immune therapy of cancer attempt to stimulate an immune response against solid tumours, infiltration of effector cells into the tumour tissue often appears to be a critical hurdle. We show that IL-23 is an important molecular link between tumour-promoting pro-inflammatory processes and the failure of the adaptive immune surveillance to infiltrate tumours.     

Somatic activation of the K-ras oncogene causes early onset lung cancer in mice

About 30% of human tumours carry ras gene mutations. Of the three genes in this family (composed of K-ras, N-ras and H-ras), K-ras is the most frequently mutated member in human tumours, including adenocarcinomas of the pancreas ( approximately 70-90% incidence), colon ( approximately 50%) and lung ( approximately 25-50%). To construct mouse tumour models involving K-ras, we used a new gene targeting procedure to create mouse strains carrying oncogenic alleles of K-ras that can be activated only on a spontaneous recombination event in the whole animal. Here we show that mice carrying these mutations were highly predisposed to a range of tumour types, predominantly early onset lung cancer. This model was further characterized by examining the effects of germline mutations in the tumour suppressor gene p53, which is known to be mutated along with K-ras in human tumours. This approach has several advantages over traditional transgenic strategies, including that it more closely recapitulates spontaneous oncogene activation as seen in human cancers.     

Tumour evolution inferred by single-cell sequencing

Genomic analysis provides insights into the role of copy number variation in disease, but most methods are not designed to resolve mixed populations of cells. In tumours, where genetic heterogeneity is common, very important information may be lost that would be useful for reconstructing evolutionary history. Here we show that with flow-sorted nuclei, whole genome amplification and next generation sequencing we can accurately quantify genomic copy number within an individual nucleus. We apply single-nucleus sequencing to investigate tumour population structure and evolution in two human breast cancer cases. Analysis of 100 single cells from a polygenomic tumour revealed three distinct clonal subpopulations that probably represent sequential clonal expansions. Additional analysis of 100 single cells from a monogenomic primary tumour and its liver metastasis indicated that a single clonal expansion formed the primary tumour and seeded the metastasis. In both primary tumours, we also identified an unexpectedly abundant subpopulation of genetically diverse 'pseudodiploid' cells that do not travel to the metastatic site. In contrast to gradual models of tumour progression, our data indicate that tumours grow by punctuated clonal expansions with few persistent intermediates.     

Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells

Transformed, oncogenic precursors, possessing both defining neural-stem-cell properties and the ability to initiate intracerebral tumours, have been identified in human brain cancers. Here we report that bone morphogenetic proteins (BMPs), amongst which BMP4 elicits the strongest effect, trigger a significant reduction in the stem-like, tumour-initiating precursors of human glioblastomas (GBMs). Transient in vitro exposure to BMP4 abolishes the capacity of transplanted GBM cells to establish intracerebral GBMs. Most importantly, in vivo delivery of BMP4 effectively blocks the tumour growth and associated mortality that occur in 100% of mice after intracerebral grafting of human GBM cells. We demonstrate that BMPs activate their cognate receptors (BMPRs) and trigger the Smad signalling cascade in cells isolated from human glioblastomas (GBMs). This is followed by a reduction in proliferation, and increased expression of markers of neural differentiation, with no effect on cell viability. The concomitant reduction in clonogenic ability, in the size of the CD133+ population and in the growth kinetics of GBM cells indicates that BMP4 reduces the tumour-initiating cell pool of GBMs. These findings show that the BMP-BMPR signalling system--which controls the activity of normal brain stem cells--may also act as a key inhibitory regulator of tumour-initiating, stem-like cells from GBMs and the results also identify BMP4 as a novel, non-cytotoxic therapeutic effector, which may be used to prevent growth and recurrence of GBMs in humans.     

Defining the mode of tumour growth by clonal analysis

Recent studies using the isolation of a subpopulation of tumour cells followed by their transplantation into immunodeficient mice provide evidence that certain tumours, including squamous skin tumours, contain cells with high clonogenic potential that have been referred to as cancer stem cells (CSCs). Until now, CSC properties have only been investigated by transplantation assays, and their existence in unperturbed tumour growth is unproven. Here we make use of clonal analysis of squamous skin tumours using genetic lineage tracing to unravel the mode of tumour growth in vivo in its native environment. To this end, we used a genetic labelling strategy that allows individual tumour cells to be marked and traced over time at different stages of tumour progression. Surprisingly, we found that the majority of labelled tumour cells in benign papilloma have only limited proliferative potential, whereas a fraction has the capacity to persist long term, giving rise to progeny that occupy a significant part of the tumour. As well as confirming the presence of two distinct proliferative cell compartments within the papilloma, mirroring the composition, hierarchy and fate behaviour of normal tissue, quantitative analysis of clonal fate data indicates that the more persistent population has stem-cell-like characteristics and cycles twice per day, whereas the second represents a slower cycling transient population that gives rise to terminally differentiated tumour cells. Such behaviour is shown to be consistent with double-labelling experiments and detailed clonal fate characteristics. By contrast, measurements of clone size and proliferative potential in invasive squamous cell carcinoma show a different pattern of behaviour, consistent with geometric expansion of a single CSC population with limited potential for terminal differentiation. This study presents the first experimental evidence for the existence of CSCs during unperturbed solid tumour growth.     

Deletion of vascular endothelial growth factor in myeloid cells accelerates tumorigenesis

Angiogenesis and the development of a vascular network are required for tumour progression, and they involve the release of angiogenic factors, including vascular endothelial growth factor (VEGF-A), from both malignant and stromal cell types. Infiltration by cells of the myeloid lineage is a hallmark of many tumours, and in many cases the macrophages in these infiltrates express VEGF-A. Here we show that the deletion of inflammatory-cell-derived VEGF-A attenuates the formation of a typical high-density vessel network, thus blocking the angiogenic switch in solid tumours in mice. Vasculature in tumours lacking myeloid-cell-derived VEGF-A was less tortuous, with increased pericyte coverage and decreased vessel length, indicating vascular normalization. In addition, loss of myeloid-derived VEGF-A decreases the phosphorylation of VEGF receptor 2 (VEGFR2) in tumours, even though overall VEGF-A levels in the tumours are unaffected. However, deletion of myeloid-cell VEGF-A resulted in an accelerated tumour progression in multiple subcutaneous isograft models and an autochthonous transgenic model of mammary tumorigenesis, with less overall tumour cell death and decreased tumour hypoxia. Furthermore, loss of myeloid-cell VEGF-A increased the susceptibility of tumours to chemotherapeutic cytotoxicity. This shows that myeloid-derived VEGF-A is essential for the tumorigenic alteration of vasculature and signalling to VEGFR2, and that these changes act to retard, not promote, tumour progression.