Solid tumours account for 90% of all cancers. Gene therapy represents a potential new modality for their treatment. Up to now, several approaches have been developed, but the most efficient ones are the viral vector based gene therapy systems. However, viral vectors suffer from several deficiencies: firstly most vectors currently in use require intratumoural injection to elicit an effect. This is far from ideal as many tumours are inaccessible and many may have already spread to other parts of the body, making them difficult to locate and inject gene therapy vectors into. Second, because of cell heterogeneity within a given cancer, the vectors do not efficiently enter and kill every cancer cell. Third, hypoxia, a prevalent characteristic feature of most solid tumours, reduces the ability of the viral vectors to function and decreases viral gene expression and production. Consequently, a proportion of the tumour is left unaffected, from which tumour regrowth occurs. Thus, cancer gene therapy has yet to realise its full potential.The facultative or obligate anaerobic bacteria have been shown to selectively colonise and regerminate in solid tumours when delivered systemically. Among them, the clostridial spores were easy to produce, stable to store and safe to use as well as having extensive oncolytic ability. However, research in animals and humans has shown that oncolysis was almost always interrupted sharply at the outer rim of the viable tumour tissue where the blood supply was sufficient. These clostridial spores, though, could serve as "Trojan horse" for cancer gene therapy. Indeed, various spores harbouring genes for cancerstatic factors, prodrug enzymes, or proteins or cytokines had endowed with additional tumour-killing capability. Furthermore, combination of these "Trojan horses" with conventional chemotherapy or radiation therapies often significantly perform better, resulting in the "cure" of solid tumours in a high percentage of animals.It is, thus, not too difficult to predict the potential outcomes for the use of clostridial spores as "Trojan horse" vectors for oncolytic therapy when compared with viral vector-mediated cancer therapy for it be replication-deficient or competent. However, to move the "Trojan horse" to a clinic, though, additional requirements need to be satisfied (i) target tumours only and not anywhere else, and (ii) be able to completely kill primary tumours as well as metastases. Current technologies are in place to achieve these goals. Gene therapy represents a potential new modality for the treatment of cancer and it is developing with a very fast pace . By the The key to a successful gene therapy is the vector system. Various vectors have been developed with unique features, including viral and non-viral based therapy systems. Although each has its own advantages and disadvantages, the replication-competent oncolytic viral vectors are the most promising amongst existing ones ,5. HowevClostridia have several advantages over others as clostridial spores specifically colonise and germinate into vegetative cells in the hypoxic regions of solid tumours, causing tumour lysis and destruction. Early trials in the 70's of non pathogenic strains in human had shown plausible safety. However, existing knowledge indicated that oncolysis was almost always interrupted sharply at the outer rim of viable tumour tissue, thus, combinational approaches have to be implemented, such as with radiofrequency therapy [C. novyi in combination with microtubule-interacting chemotherapeutic agents, including vinorelbine and docetaxel and demonstrated very promising results. A phase 1 trial of the combined approach in patients is in progress [Of all cancer diagnosed, 90% of these are solid tumours. Recent understanding of the unique pathology of solid tumours has shed light on the disappointing nature of these new therapies and now demands rational and innovative design of vectors. All solid tumours, when they grow more than 2 mm diameter in size, undergo angiogenesis that results in biological changes and adaptive metabolisms, i.e.: formation of defective vessels, appearance of hypoxic areas, and emergence of heterogeneous tumour cell population . This mi therapy ,9. A newprogress .The intrinsic property of tumour-targeted colonisation of clostridia enables them to serve as "Trojan horses" for the delivery of genes for cancer therapy. Indeed, clostridial spores that were genetically manipulated to harbour genes for cancerstatic factors, prodrug converting enzymes, or cytokines to improve their innate oncolytic activity have been developed, including our work and others ,12. FurtThis review briefly describes the viral vectors, including the replication defective vectors, the targeted vectors and replication-competent oncolytic vectors, and their use in cancer gene therapy as well as their advantages and disadvantages. Subsequently, we will focus on the development of clostridial spores as "Trojan horse" vectors for cancer therapy, the mechanisms involved, and the foreseeable promises and problems when compared with existing viral vector systems.in vivo use of the vector system. However, studies in animal models have shown that poor vector penetration is always a problem in vivo. MoMLV vector rarely travelled away from the injection sites [i.c.) injection of replication-competent MoMLV [Murine Moloney leukaemia virus (MoMLV)-based retroviral vector is one of the earliest systems that were developed for gene therapy. The vector has unique ability to transduce dividing cells . Tumour on sites . Due to nt MoMLV . Viral eOverall, more than 23% of all gene therapy trials in patients for various diseases have used a replication-defective or competent MoMLV vector system. Existing studies in animal experiments have shown that the vector system was relatively safe and non-toxic. The case against retroviral vector systems is potential problems related to activation of cellular oncogenes and inactivation of tumour suppressor genes by insertional mutagenesis. This was true in the case of using MoMLV to transducer bone marrow stem cells for the therapy of severe combined immune deficiency syndrome (SCIDs), 4 out of 11 treated children have now developed leukaemia . In addiin vitro and in vivo [8 plaque forming unit (PFU)/ml of virus in the plasma), and being able to efficiently integrate into chromosomes of non-dividing and terminally differentiated cells. Most of the lentiviral vectors were pseudotyped with a glycoprotein from the vesicular stomatitis virus (VSV), VSV-G, as it provided not only a broad tropism, but also physical strength that enabled concentration by centrifugation.Lentiviral vector is a new type, more complicated retroviral vectors, which are primarily based on Human or Bovine Immunodeficiency Virus. They have all the unique features of MoMLV and have been shown to transduce post mitotic cells  in vivo . Studies in vivo . Thus, s in vivo ,21. JDV Adenovirus vector is the most commonly studied and most widely used system in cancer gene therapy. They are of particular utility for cancer gene therapy applications, where temporary gene expression is acceptable or even beneficial. The currently employed AVs in clinical trials are all based on serotype 5. AV can be produced at high titre and has demonstrated efficient gene transfer to various types of cancer cells . Two AVsSeveral other adenoviruses, based on canine, porcine, bovine, ovine and avian have all been developed. The ovine AV is based on serotype 7 and developed in Australia. Preclinical testing on prostate cancer in animal models has shown therapeutic efficacy .in vivo recombination. With regard to AV-mediated cancer treatment, high-level tumour transduction remains a key developmental hurdle. To this end, AV vectors possessing infectivity enhancement and targeting capabilities should be evaluated in the most stringent model systems possible. Advanced AV-based vectors with imaging, targeting and therapeutic capabilities have yet to be fully realized; however, the feasibilities leading to this accomplishment are within close reach [AVs contain many viral genes encoding major proteins that elicit a strong host immune response. Of particular concern is the development of cytotoxic T lymphocytes that lyse cells expressing the recombinant genes. Newer generations of AV vector were designed to overcome some of these problems and the initial results were encouraging. New techniques involved in removing the recombinant viral genes and transfecting the non-recombinant plasmid with a helper virus and then separating the helper virus with sedimentation techniques were developed. Improvements in helper virus have also been trialled that reduces "floxed" helper virus production 1000-fold, but this method still has a 1% wide type (WT) contamination thus still allowing the possibility of se reach .AAV-based vectors have been shown to be non-toxic and undergo widespread cellular uptake in preclinical evaluation . A recenVectors based on herpesviruses are well-developed and have progressed to clinical trials. As with other viral vector, replication defective vectors did not show much of use. The first replication competent vector was based on a mutant strain, where the vectors are deleted of the main neurovirulence gene r34.5, which restricted its ability to replicate in adult central nervous system and to form latency. However, later study showed that the mutant strain that had the deletion of the r34.5 gene also reduced the capacity of replication inside tumour cells . The newPre-existing immunity may pose a problem that limits the clinical efficacy of herpesvirus-based vectors. The immunity prevented the transduction of peripheral organs and also caused liver toxicity. However, a recent mutant strain-secreting cytokine granule macrophage colony stimulatory factor (GM-CSF) or IL-12 was shown to be effective in liver cancer therapy in a murine model which likely involves both direct viral oncolysis and actions of specific immune effector cells .Semliki Forest virus (SFV) subgenomic replicons (i.e. non toxic replication) have been developed that allow stable expression of a required gene e.g. beta-galactosidase  in mammalian cell lines. Expression remained high (approximately 150 pg per cell) throughout cell passages .mariner fish element [Since construction of the Sleeping Beauty transposon from defective copies of a Tc1/ element , new verWhile efforts have been focused on the continuing refinement of various vector systems, several obstacles remain, primarily the low efficiency of gene delivery into target tumour cells. The vascular endothelial wall is a significant physical barrier prohibiting access of systemically administered vectors to the tumour cell. To overcome this obstacle, strategies are currently being developed to take advantage of transcytosis pathways through the endothelium. An AV vector targeted to the transcytosing transferrin receptor pathway, using the bifunctional adapter molecule had been constructed . The tra18F-penciclovir, providing an imaging system for viral replication that can directly be translated for clinical applications. Interestingly, HSV TK is an enzyme that has utility in so-called suicide gene therapy, in which the expressed enzyme converts a substrate such as ganciclovir to its phosphorylated metabolite, which can then be further phosphorylated by cellular kinases to a toxic metabolite, causing cell death [Recently, a concept of multifunctional particles (MFPs) based viral vectors has emerged. The idea incorporated viral vectors' tumour targeting, imaging and amplifying tumour killing capacities. AAV has been developed as MFP, by virtue of genetic capsid modifications, to incorporate additional functionalities, such as modified fibres, combined with imaging motifs on the pIX protein, to simultaneously target tumour cells while monitoring viral replication and spread. HSV TK has been incorporated at pIX site of the AAV capsid. This enzyme is compatible with available PET imaging ligands such as ll death . Also, tNanotechnology has also been introduced recently in the context of MFP. This is defined as the development of devices of 100 nm or smaller, having unique properties due to their scale. The devices that are being developed generally incorporate inorganic or biological material. In this regard, the coupling of inorganic nano-scale materials to targeted AV vectors has much potential. For example, magnetic nano-particles have recently received much attention due to their potential application in clinical cancer treatment; targeted drug delivery and magnetic resonance imaging (MRI) contrast agents . Howeverwt-p53 in cancer cells through gene transfer techniques restores p53 function and triggers apoptosis leading to tumour cell destruction [wt-p53 following gene transfer.The p53 tumour suppressor gene has received a great deal of attention as a cancer therapeutic strategy due to the important role it plays in maintaining the integrity of the genome. It is involved in cell cycle regulation, DNA repair, and apoptosis, essentially controlling the integrity of the genome. Following exposure to DNA damaging agents, p53 activation results in cell cycle arrest, allowing for DNA repair or triggers cellular apoptosis if the damage is irreparable. Thus, p53 mutations in cancer allows for unregulated cellular proliferation in the face of genetic mutations and accumulation of genetic errors contributing to the malignant phenotype and genomic instability of cancer cells. Preclinical studies have demonstrated that replacement of truction ,37. The p53 gene repair clinical trials have been initiated. These trials have used different vector systems for gene transfer (retrovirus and adenovirus), different routes of vector delivery , and have focused on different subtypes of lung cancer . The first phase I clinical trial of such an approach was conducted by Roth et al. at the M.D. Anderson Cancer Centre [p53 via CT-guided or endobronchial injections. Effective gene transfer was demonstrated in biopsied tumours following injection and some degree of tumour regression of the injected lesion was seen in three subjects providing proof-of-concept for this gene therapy approach. All subsequent trials have utilized adenoviral vectors for gene transfer since such vectors are relatively easy to manufacture at large scale, can be produced at higher viral titres, and have the ability to transduce both dividing and non-dividing cells. Three adenoviral p53 (Ad-p53) single agent trials have been performed as well as three Ad-p53 combination trials. Two of the single agent trials were performed in advanced NSCLC using either single or multiple vector injections [p53 gene transfer as the sole treatment modality whereas more recent trials have combined p53 gene transfer with other cancer therapies, notably chemotherapy or radiation, as part of a combined modality treatment approach.Based on these preclinical studies a number of r Centre . In thisjections . These tjections . Neverthjections , or radip53 gene transfer approaches described above, preclinical data with adenoviral HSV TK gene transfer (AdHSV TK) followed by ganciclovir exposure results in a bystander effect in which neighbouring, non-transduced cells are also being killed, presumably due to transfer of toxic metabolites from the transduced cells as well as induction of a generalized immune response [This is also one of the well studied strategies and is based on the delivery of a "suicide-toxin producing enzyme" gene not normally found in mammalian cells to tumour cells that allows for selective sensitivity to a systemically administered pro-drug. One such suicide gene is the HSV TK gene that codes for an enzyme that converts the normally nontoxic nucleoside analogue, ganciclovir, in subsequent steps into a toxic compound that leads to tumour cell death. Like the adenoviral response . Preclinresponse . Gene trAt present, the hurdle to clinical use of this strategy is the low efficiency of gene transfer. To overcome the problem, we also developed a novel way of using a unique peptide to shuttle the HSV TK gene to neighbouring cells . Howeverex vivo and in vivo approaches. Early studies of adoptive transfer of ex vivo expanded tumour infiltrating lymphocytes (TIL) demonstrated responses in melanoma and renal cell cancer but activity in other solid tumours was limited [Several genetic strategies have been employed to enhance the immunogenicity of tumours with a goal of inducing immune-mediated tumour destruction. Unlike the gene repair and suicide gene transfer studies described above, immunogene therapies have the theoretical advantage of inducing a systemic anti-tumour response associated with immunologic memory. Such a response potentially allows for treatment of disseminated disease and a prolonged anti-tumour effect that persists beyond the immediate treatment period. Immunogene therapy strategies involve both  limited . Systemiex vivo approach using the lentiviral vector-mediated transfer of the tumour antigen gene into dendritic cells (DCs) cells. Therapeutic effects were demonstrated in up to 85% of the subjects [We have developed an subjects .Raf gene to angiogenic blood vessels using αvβ3-targeted liposomes. The integrin, αvβ3, is preferentially expressed in the angiogenic endothelium and contributes to viral internalization making it a good targeting molecule for anti-angiogenic gene therapy strategies. Raf is a cellular signalling molecule that plays an important role in neovascularization. Mice lacking Raf die early in development with vascular defects and a mutant form of Raf was shown to block angiogenesis in response to pro-angiogeneic factors in vitro. Systemic injection of targeted liposomes conjugated to a mutant Raf gene into mice with pre-established lung and liver metastases from colon carcinoma demonstrated predominant tumour endothelial cell uptake, tumour endothelial cell apoptosis, and pronounced tumour regressions.One of the features of the malignant tumours was the increased vasculature. Therefore, targeting tumour vasculature rather than the tumour cell itself as a treatment for cancer has gained increasing interest in recent years. A number of inhibitors of angiogenesis . VEGFR2 has relatively restricted expression on endothelial cells and is upregulated in proliferating tumour neovasculature. An orally available DNA vaccine encoding murine FLK-1 was shown to suppress angiogenesis in tumour vasculature, protect mice from lethal challenges with melanoma, colon, and lung carcinoma cells, and reduce the growth of established metastases .in the growth inhibition of NSCLC cells. A further study is underway to demonstrate the in vivo efficacy of EGFR silencing in animal models [One of the newer technologies in cancer gene therapy involves the silencing of genes in cancer cells that regulate tumour cell growth and proliferation. We have developed a double stranded RNA mediated silencing of the epidermal growth factor receptor (EGFR). In vitro studies have demonstrated effective silencing of the EGFR and resulted l models "C. histolyticum into mouse sarcoma caused oncolysis (liquefaction) and tumour regression [i.v. with spores of C. tetani, the causative agent of tetanus. Injected non-tumour bearing animals remained healthy. However, tumour bearing mice died within 48 h because of C. tetani colonisation and tetanus production. This provided evidence that the C. tetani were able to germinate/replicate selectively in the anaerobic environment found within tumours, and released their toxins systemically [C. butyricum M-55 has been isolated [C. oncolyticum and taxonomic studies have now clearly established that it is a C. sporogenes strain (ATCC13732). This is a proteolytic species causing liquefaction of colonised tumours. This was later verified by testing more isolates.It is obvious that a major step towards the development of an effective cancer therapy will be to construct a vector that targets the tumour alone, and is capable of spreading to and throughout the tumour found in tissues. Clostridial spores fit into this equation very well. Clostridia are strictly anaerobic. They are gram-positive, rod-shaped, and form spores under unfavourable conditions. There are about 80 species and several of these have been tested in solid tumours. All known species require anaerobic conditions to grow but do vary in their oxygen tolerance and their biochemical profile. Clostridial spores have been administered intravenously and showed a distinct advantage for use in cancer therapy as they are easy to produce and store. Germination of spores will only occur when they encounter the requisite anaerobic conditions. Spontaneous colonization of tumours in cancer patients and the apparent selectivity of Clostridia for tumours were noticed more than 50 years ago. The first experiment in 1947 showed that direct injection of spores of gression . Later eemically . Obviousisolated . M-55 waC. beijerinckii NCIMB8052 spores administered intravenously to EMT6 tumour-bearing mice germinated in the necrotic tumour regions while the oxygenated tumour areas remained free of spores [C. beijerinckii ATCC17778, C. acetobutylicum DSM792 (= ATCC824) or C. acetobutylicum NI-4082 (reclassified as C. saccharoperbutylacetonicum) showed tumour colonisation without complete tumour lysis [Saccharolytic clostridia, such as f spores . Equallyur lysis .C. sporogenes was the first Clostridium to be gene modified and this was performed with the E. coli Colicin E3 gene. Colicin E3 encodes a bacteriocin shown to have canceriostatic properties [E. coli cytosine deaminase (CD) into C. sporogenes NCIMB10696 by electroporation [C. sporogenes has a great capacity to colonise the tumour. At least 10e8 CFU/g of tumour was obtained following the intravenous injection of the spores  or C. acetobutylicum NI4082 (reclassified as C. saccharoperbutylacetonicum) and C. butyricum are non-pathogenic and their development has been industry funded. Therapeutic genes, encoding the cytokine tumour necrosis factor alpha (TNF-α), CD or nitroreductase (NTR) have been introduced into these strains [C. acetobutylicum using strain-specific electroporation protocols, CD expression was monitored in lysates and supernatants of early logarithmic growth phase cultures of recombinant C. acetobutylicum (pKNT19closcodA) [C. acetobutylicum strains NI4082 and DSM792 engineered to produce cytosine deaminase were able to express and secrete this enzyme at the tumour site [C. acetobutylicum, but not in control animals. Animals, concomitantly treated with antivascular chemical agent, CombreAp, showed higher incidence of CD-positive tumours (100 versus 58%). Moreover, the level of active CD in these tumour specimens was considerably higher (mean conversion efficiency of 5-FC to 5-FU ~11%) as compared to tumours not treated with the vascular targeting drug (mean conversion efficiency of 5-FC to 5-FU ~11%) when compared to untreated tumours (mean conversion efficiency of 5-FC to 5-FU ~3%) [in vivo, there was a consistent lack of significant tumour regression observed. Factors that may have contributed to this lack of efficacy include a low level of bacterial colonisation of the tumour or insufficient recombinant gene expression and secretion at the tumour site [Clostridial strains C. sporogenes or C. novyi-NT. Recombinant C. sporogenes and C. novyi-NT overexpressing NTR showed significant in vivo anti-tumour effects [Saccharolytic Clostridia strains including  strains ,59. FollloscodA) . A consiour site ,59. Funcour site . Recent  effects  when useAt present, there are various gene therapy vector systems under development against cancer. However, due to the complexity of the solid tumours involving angiogenesis, hypoxia, stromal cell, tumour cell heterogeneity and the emergence of de-differentiated stem cells, none of the existing vectors are holding any real promises. The clostridial spore-based vector system is not infectious, and has gained renewed interest, because of the following true advantages.C. novyi-NT has demonstrated that the strain was unable to colonise artificially created infarcted heart where the level of hypoxia was inadequate to support the replication of the Clostridia. Early trials of non-pathogenic Clostridia strains in patients have demonstrated safety. In the unlikely event of an adverse effect, clostridia can be eliminated from the blood stream with the use of readily available antibiotics such as metronidazole which showed total spore clearance from the blood stream after 9 days of treatment [Safety is always a concern when live vector systems are used for human gene therapy. Some of the hurdles of using viral vectors include: (1) whether the vector is sufficiently targeted to tumour alone; (2) whether the vector expresses low levels of viral genes that may lead to increased toxicity and immunogenicity ; (3) posreatment .The biological properties of virus-based vectors, in particular the ability to enter and replicate  within a tumour cell and then spread from cell to cell are highly relevant for effective cancer therapy. However, recent understanding of tumour pathology has revealed that several features of the tumour environment may not be conducive for viral replication ,66. HypoAll of the viral vector systems need sophisticated cell culture systems, expensive culture media, rounds of filtrations and purifications and dedicated centrifugation and storages. On the contrary, clostridial spores can be easily and inexpensively produced from anaerobic bacterial culture. There are only a few steps involved and the spores, once produced can be stored at room temperature for at least 3–6 months.While most viral vectors have to be intratumourally injected, intravenous injection of resuspended clostridial spores are possible and sufficient as they will be leaked out of the incomplete vessels in the solid tumour, thus specifically targeting to and colonising the hypoxic regions of the tumours.Solid tumours comprise not only malignant cells, but also extracellular matrix and many other non-malignant cell types, including stromal cells such as fibroblasts, endothelial cells and inflammatory cells. The mechanisms of clostridial vector-mediated tumour killing consist of several aspects: one is from its transgene that encodes prodrug converting enzymes for suicide-gene therapy or cytokines for immuno-gene therapy. These are essentially the same as the viral vectors. However, there is another side of the tumour killing effect that is resulting from the consequences of an innate antitumour effect of the clostridial strain due to production of hydrolytic enzymes including proteases, lipases, and nuclease. Furthermore, there is also a nutrients competition between the clostridia and cells surrounding them , where the clostridia multiplied much faster than the mammalian cells. The cumulative multiplications and the combined events of energy and substance metabolism effectively depleted the limited nutrient source and deprive the tumour cells, causing starvation and death. More recently, there were observations that indicate the germination of the clostridial spores, the transformation from spores to vegetative rods, and the continue multiplications of the vegetative rods inside the tumour activated the immune system, assisting the antitumour effects . These tWhile viral vectors need access to viable target cells and their cellular machinery to achieve transgene delivery and expression, this goal is often difficult to fulfil as some tumour cells are not viable at the time of gene delivery. Furthermore, none of the existing vector systems efficiently transfer genes to every tumour cell which subsequently allows for tumour regrowth. On the other hand clostridial spore replication is not tumour cell dependent and occurs via rod multiplication extra-cellularlly. Furthermore, the tumour killing mechanism of clostridial spores may operate independently of the requirement for gene transfer. Without the requirement for gene integration into the host cell genome removes the possibility of insertional mutagenesis when using Clostridia. Therefore, Clostridia may show tumour killing irrespective of the tumour cell heterogeneity found within the tumour environment.One of the primary limitations of most viral vectors has been the small size of the virion, which only permits the packaging of very limited sizes  of exogenous DNA that includes the promoter, the polyadenylation signal and any other enhancer elements that might be desired. However, for clostridia size limitations are far less restricted, not only because the plasmids used can harbour much larger DNA fragments, but in case the foreign gene is integrated in the host chromosome there is in fact unlimited capacity for insertion of therapeutic genes, forecasting the promising future for the development of ever powerful vectors.The unique pathophysiology of solid tumours presents a huge problem for the conventional therapies. Thus, the outcomes of current therapies are so far disappointing. Several new approaches aiming at developing effective treatments are on the horizon. These include a variety of virus-based therapy systems -71. AmonC. novyi-NT) with an antimicrotubuli agent has been initiated [The strictly anaerobic clostridia, on the other hand, have been shown to selectively colonise in solid tumours when delivered systemically and has resulted in high percentage of "cures" of experimental tumours. A phase I clinical trial combining spores of a non toxic strain ; Dendritic cells (DCs); Colony forming unit (CFU); Plaque forming unit (PFU); Epidermal growth factor receptor (EGFR); Herpes simplex thymidine kinase (HSV TK); Interleukin-2 (IL-2); Granule macrophage colony stimulatory factor (GM-CSF); Human Immunodeficiency Virus (HIV); Jembrana Disease virus (JDV); Magnetic resonance imaging (MRI); Murine Moloney leukaemia virus (MoMLV); β-galactosidase ; Multifunctional particles (MFPs); Nitroreductaes (NTR); Non small cell lung cancer (NSCLC); Semliki Forest virus (SFV); Severe combined immunodeficiency syndrome (SCIDs); tumour infiltrating lymphocytes (TIL); Tumour necrosis factor (TNF); Vascular endothelial growth factor (VEGF); Vesicular stomatitis virus (VSV); Wide type (WT).Adenoviral vector (AV); Adeno-Associated Virus (AAV); The author(s) declare that they have no competing interests.All authors participated in the production of the manuscript together and have read and approved the final manuscript.


The fluorinated pyrimidine nucleoside, 5′-deoxy-5-fluorouridine (5′-dFUrd) has been shown to effectively attenuate the progress of cachexia in the murine adenocarcinomas MAC16 and colon 26 as well as in the human uterine cervical carcinoma xenograft, Yumoto. Although concomitant inhibition of tumour growth was observed in all three models this was not sufficient to account for the preservation of body weight. An attempt has been made to correlate the anti-cachectic activity of 5′-dFUrd with the presence of a tumour produced proteolysis-inducing factor (PIF), thought to be responsible for the development of cachexia in the MAC16 model. Two variants of colon 26 adenocarcinoma were employed, clone 20 which produces profound cachexia, and clone 5 which produces no change in body weight in recipient animals. Mice bearing the colon 26, clone 20 variant showed evidence for the presence of PIF in tumour, serum and urine, while there was no evidence for the presence of PIF in tumour or body fluids of mice bearing the clone 5 tumours. Treatment of animals bearing the clone 20 variant with 5′-dF Urd led to the disappearance of PIF from the tumour, serum and urine concomitant with the attenuation of the development of cachexia. The human cervical carcinoma, Yumoto, which also induced cachexia in recipiant animals, showed expression of PIF in tumour, serum and urine in control and vehicle-treated mice, but was absent in mice treated with 5′-dFUrd. Thus in these experimental models cachexia appears to be correlated with the presence of PIF. © 2000 Cancer Research Campaign


Direct evidence of tumour seeding in distant organs at the time of surgery for gastric cancer is not available. An immunocytochemical assay for epithelial cytokeratin protein may fill this gap since it is a feature of epithelial cells that would not normally be present in bone marrow. The bone marrow of 46 patients with primary gastric cancer was examined for tumour cells, using immunocytochemical techniques and antibody reacting with cytokeratin, a component of the intracytoplasmic network of intermediate filaments. The monoclonal antibody CK2 recognises a single cytokeratin polypeptide (human cytokeratin no. 18) commonly present in epithelial cells. The expression of tumour-suppressor genes p53 and RB for the primary lesion was also determined using the monoclonal antibodies PAb 1801 and 3H9 respectively, and the proliferating activity was determined by the Ki-67 antigen labelling index for MIB-1 antibody staining. Of these 46 patients, 15 (32.6%) presented with cytokeratin-positive cells at the time of primary surgery. The positive findings were related to the undifferentiated tissue type and to the prominent depth of invasion, but not to other clinicopathological factors. In 2 of 15 (13.3%) patients, the depth of invasion was limited to the mucosa. The metastatic potential to bone marrow did not relate to expressions of p53 and RB genes, or to the proliferating activity of MIB-1 staining for the primary lesion of gastric cancer. As tumour cells in bone marrow are indicative of the general disseminative capability of an individual tumour, this technique may be useful for identifying patients at high risk of metastasis from a gastric tumour.


Angiogenesis is an essential requirement for tumour growth and metastasis and is regulated by a complex network of factors produced by both stromal cells and neoplastic cells within solid tumours. The cytokine tumour necrosis factor alpha  and the enzyme thymidine phosphorylase (TP) are two factors known to promote tumour angiogenesis. We have demonstrated recently that high numbers of tumour-associated macrophages (TAMs) are significantly associated with increased tumour angiogenesis and poor prognosis in invasive carcinoma of the breast. We have also shown that TAMs are a major source of TNF-alpha in invasive breast carcinomas, and that macrophage-like stromal cells as well as tumour cells synthesize TP in such tumours. However, little is known of the factors that regulate the production or activity of these factors in the tumour microenvironment. As TNF-alpha has been shown to up-regulate TP expression in tumour cells in vitro we performed an immunohistochemical study to investigate the possibility that TNF-alpha may be involved in the regulation of TP expression by malignant breast epithelial cells in vivo. To do this, we used a cocktail of non-neutralizing monoclonal anti-TNF-alpha antibodies to visualize both TNF-alpha-expressing macrophages and TNF-alpha bound to its receptors on tumour cells and endothelial cells in a series of 93 invasive carcinomas of the breast. A semiquantitative grading system was then used to compare these staining patterns with that for TP in the same biopsies. TNF-alpha immunoreactivity was also compared with various important tumour variables known to relate to outcome in this disease , as well as relapse-free and overall survival data for these patients. Our data show significant positive correlations between TNF-alpha bound to its receptors on tumour cells and: (1) TP protein production by tumour cells, and (2) axillary lymph node status (i.e. metastasis). These results suggest that tumour cell responsiveness to TNF-alpha produced by neighbouring TAMs may play a part in the regulation of TP expression by tumour cells as well as their metastatic behaviour. This may explain, in part, the relationship between increased macrophage infiltration and angiogenesis in breast cancer, and further supports the contention that TAMs may represent an important target for future anti-angiogenic therapies.


We present a case of a symplastic or "bizarre" leiomyoma of the scrotum. Isolated cases of leiomyomas have been reported arising from the renal pelvis, bladder, spermatic cord, epididymis, prostate as well as the glans penis. However such mesenchymal lesions of the scrotum are very rare.Macroscopically the tumour was a well-circumscribed grey-white lesion 8.5 cm in size. Because of its peculiar histological characteristics this tumour was assigned as a symplastic or bizarre leiomyoma of the scrotum.We present this unusual tumour and highlight some important diagnostic and treatment pitfalls related to this rare tumour. This case demonstrates that leiomyomas should be considered in the differential diagnosis of scrotal tumours. A 59-year old Caucasian man of average height and build presented with an 18-year history of a slowly enlarging, painless hard mass arising from the lower pole of his right scrotum (figure A scrotal ultrasound confirmed normal testes and cord structures but showed a bizarre solid mass with a heterogeneous disorganised pattern with poor vascularity suggesting a mesenchymal tumour arising from the scrotal wall. This mass was excised under a general anaesthetic. The patient made a good recovery post-operatively. Macroscopically the tumour was a well-circumscribed grey-white lesion 8.5 cm in size. Because of its peculiar histological characteristics this tumour was assigned as a symplastic or bizarre leiomyoma of the scrotum.Conventional leiomyomas may originate from any anatomic location of smooth muscle in the genitourinary system. IsolateSymplastic leiomyomas are rarely reported lesions in the medical literature -12 A medOn gross sections these tumours appear white-grey and are well circumscribed and encapsulated. On high power images these neoplasms are characterised by interlacing bundles of spindle shaped muscle cells with pleomorphic nuclei and occasional nuclear inclusions Figure . The musIt is important to emphasize that despite their histological characteristics on presentation, compatible with malignancy, these tumours have a benign course not any different from conventional leiomyomas even when they are larger in size than those reported in the literature. In contrast to scrotal leiomyosarcomas, scrotal leiomyomas with bizarre nuclei are not hypercellular, and they lack mitotic activity.Ultrasound scan appears to be the investigation of choice in the pre-operative assessment of these tumours ,9 and shThis case report highlights some important diagnostic and treatment issues related to this rare tumour. Histologically they behave differently to both conventional leiomyomas as well as leiomyosarcomas. Their behaviour is benign in nature although it is not clear if they can recur locally. Hence follow-up of these patients is advised. This report highlights the clinicopathological characteristics of the scrotal bizarre leiomyoma in order to increase our understanding, and avoid the possibility of erroneous diagnosis and treatment. It is very important to distinguish these bizarre or symplastic leiomyomas from leiomyosarcomas to avoid unnecessary treatment.Written consent was obtained from the patient to publish this manuscript and accompanying images. A copy of this consent is available for review by the editor of this journal.The authors declare that they have no competing interests.JM and SV did the literature search and wrote the draft article. PA was responsible for the histology slide and histological input. TWC revised the article for intellectual content.


Tumour necrosis factor-alpha  reduced the interstitial fluid pressure (IFP) to 54-64% (P < 0.05) and the mean arterial blood pressure (MABP) to 70% (P < 0.01) of control values after 5 h in three human melanoma tumour lines transplanted to nude mice.


In addition, we assessed TBF stasis after i.v. and intra-arterial 10 mg kg−1 AC7700 administration in an LY80-induced kidney tumour. To determine why the TBF stasis is irreversible, we observed AC7700-induced changes in host arterioles and the tumour vascular network of the Sato lung carcinoma using a vital microscopic rat transparent chamber. Since an increase in tumour interstitial fluid pressure brings about a decrease in TBF, we also measured 10 mg kg−1 AC7700-induced changes in this pressure. The sensitivity of the blood flow response after intratumoral application of AC7700 was markedly higher in normal vessels relative to tumour vessels. Intra-arterial administration of AC7700 did not have stronger effects on TBF stasis than did i.v. administration. Intravital microscopy showed that AC7700 induced a powerful and long-lasting constriction of host arterioles, so that complete stasis of blood flow occurred in downstream vessels, which supplied blood to tumours. Owing to this stasis, the lumens of numerous tumour vessels narrowed or completely disappeared, and numerous erythrocytes stagnated in drainage vessels of the tumour vascular network. Haemolysis of these erythrocytes occurred after 2–3 h, resulting in complete thrombosis. There was no indication of reperfusion in vessels showing haemolysis. This haemolysis is thought to be the main cause for the irreversibility of TBF stasis. Since the tumour interstitial fluid pressure decreased after i.v. AC7700 administration, the possibility of stasis of TBF being caused by tumour vascular compression was excluded. All these results strongly suggest that the main target of AC7700 is host arterioles and that the stasis of TBF induced by AC7700 is not triggered by a direct action of the drug on tumour vessels.We previously reported that a novel combretastatin A-4 derivative, AC7700, has remarkable antitumour effects because of an irreversible stasis of tumour blood flow (TBF) and subsequent loss of nutrient supply to tumour tissue. Since early 2002, under the new designation AVE8062, AC7700 has undergone clinical trials in Europe and the US. Questions remain, however, concerning how AC7700 blocks TBF and why the TBF stasis does not recover. In this study, using a rat tumour LY80, a variant of Yoshida sarcoma, we examined whether TBF cessation after AC7700 administration is due to a direct action of the agent on tumour blood vessels. We constructed electrodes that can drop a small quantity of the drug solution directly at the site of blood flow measurement and inserted them subcutaneously and into the tumour. We compared the blood flow responses of normal vessels and tumour vessels after administration of 10- It was first isolated from the bark of the South African bush willow by in vivo (Combretastatin A-4 (CA-4) is an antimitotic agent with et al, unpublished data) and antitumour effects  to determine whether the cause of TBF stasis induced by AC7700 is the result of a direct action of this agent on tumour vessels; (b) to determine by means of intravital microscopy why the drug-induced stasis is irreversible; (c) to determine whether AC7700, like CA-4-P, can increase tumour vessel permeability and thereby increase tumour interstitial fluid pressure (TIFP); and (d) to construct a self-consistent model of the various phenomena observed after AC7700 administration and thereby elucidate the microcirculatory mechanisms that lead to irreversible TBF stasis and subsequent necrosis.Male Donryu rats , 8–10 weeks old and with an average weight of 250–300 g, were used for all experiments. Rats were bred and maintained in a ventilated, temperature-controlled (24±1°C), specific pathogen-free environment on a bed of wood shavings with food and water freely available and a 12-h light–dark cycle. They were usually housed two or three per cage. The rats equipped with transparent chambers for microscopic observations (see below) and the rats fitted with diffusion chambers for measurements of TIFP (see below) were caged singly.Tumour cell lines included LY80, which is a variant of the Yoshida sarcoma, and Sato lung carcinoma (SLC), which is an undifferentiated lung carcinoma. In our laboratory, LY80 and SLC are maintained by successive i.p. and s.c. transplantations, respectively. Although SLC and LY80 showed almost the same growth potential in the transparent chamber and showed almost the same reaction after AC7700 administration, we chose SLC rather than LY80 for vital microscopic observations. We did so because when SLC is growing in transparent chambers, demarcation between the edge of the growing tumour and the normal tissue is clear  and Notification (No. 6) issued by the Japanese Government. The ethical guidelines that were followed meet the standards required by the UKCCCR . For the investigation of topical application of the drug to the tumour tissue, and for the experiment measuring TIFP using the wick-in-needle method, the tumour cell suspension was injected s.c. into the back of each rat. For measuring TIFP by the diffusion chamber method, the chamber was first implanted in the back of the rat and then a suspension (0.1 ml) containing 2 × 106 cells was injected around the chamber. For the experiment investigating the effect of intra-arterial (i.a.) AC7700 injection, tumour cells were implanted into the kidney. Recipient rats were anaesthetised and placed in the left lateral decubitus position, and a vertical incision was made in the right flank through the skin and peritoneum to expose the lateral aspect of the kidney. The tumour cells (2 × 106 cells in 10 μl) were implanted into the renal parenchyma using a 50-μl graduated syringe  and a 27-gauge syringe needle . The site of injection was 2 mm below the renal capsule. The hole made by the needle was sealed with synthetic resin glue . After the injection, the incision wound was closed in one layer with thread, and the animals were allowed to recover.LY80 cells growing in ascites of a donor rat were collected, suspended in pH 7.4 phosphate-buffered saline, and adjusted to a concentration of 2 × 10−1 using an infusion pump . In all experiments of the present study, the AC7700 administration was completed within 2 min. The solution volume administered was 1 ml kg−1. Fluorescein isothiocyanate dextran (molecular weight (m.w.) 4400 Da)  was used as a fluorescent contrast material. Experiments were performed with the animals anaesthetised in a temperature-controlled (24±1°C) box fitted with a suction duct. Both pentobarbital sodium salt  and enflurane  were used simultaneously for anaesthesia. The pentobarbital powder was solved in distilled water  to give a concentration of 50 mg ml−1 and was administered intramuscularly (i.m.) at a dose of 30 mg kg−1 10 min before the experiment. When the experiment continued for more than 2 h, supplemental doses (15 mg kg−1 i.m.) were given at 90-min intervals to maintain immobilisation. Enflurane concentration was maintained at 1% in the inhaled carrier gas, which was administered at a rate of 1 l min−1 by means of an anaesthetic apparatus for small laboratory animals (AC7700 (AVE8062), one of the combretastatin derivatives, was synthesised and provided by Ajinomoto Pharmaceutical Research Laboratories, Kawasaki, Japan. The AC7700 powder was dissolved in 0.9% NaCl solution immediately before use. The AC7700 solution was usually injected into the tail vein at a rate of 0.15 ml min−1), the blood flow value (in ml min−1 (100 g tissue)−1) in a small region was calculated from the half-life of the clearance curve obtained. A tissue blood flow meter with two separate amplifiers  was used. Two 80-μm-diameter hydrogen electrodes  and two rod-type Ag/AgCl reference electrodes  were usually used for each rat. The reference electrodes were inserted between the skin and musculature in the caudal region. In the experiment measuring TIFP, one electrode was used to monitor TBF. In the experiment measuring TBF change after topical application of AC7700, a special electrode (see below) was used.The tumour blood flow was measured using the hydrogen clearance method , 2002 thThe tumour blood flow was measured 7–10 days after tumour implantation. For measuring TBF in the kidney tumour, a vertical incision was carefully made in the right flank through the skin and peritoneum, similar to the incision for implantation of the tumour, and electrodes were inserted into the solid tumour growing in the renal parenchyma, to a depth of 2 mm from the tumour surface. Throughout the experiment, rats were placed prone on a heated stage at 34°C and kept in the same position. The rectal temperature monitored with a thermistor for small animals  was 33.5–35.5°C and the condition was maintained.The mean arterial blood pressure (MABP) was monitored in all rats in which TBF was measured. The mean arterial blood pressure was measured via a catheter  inserted into the right femoral artery. Pressure in the catheter was recorded with a pressure transducer , the output of which was fed into an amplifier  adapted for such a measurement.μm). The sensitive part of the electrode is the 1-mm tip, which detects hydrogen gas. At 10 mm from the tip, the wire is passed through a pinhole made in the sidewall of the tube. The pinhole is packed with silicone rubber. With this electrode, we were able to measure the dose–response relationship for AC7700.To investigate whether tumour vessels or normal vessels are more sensitive to AC7700, we constructed a new type of wire electrode for use in the hydrogen clearance method. From the tip of the wire, a section 1–10 mm long is enveloped by a thin polyethylene tube . Through this tube, 10 μl of AC7700 solution  or 10 μl of 0.9% NaCl solution was dropped directly on the region at which blood flow would be measured.For tumours, the tip of the electrode was placed at a depth of 4 mm below the tumour surface. For normal tissue, the electrode was introduced subcutaneously into the animal's back via a syringe needle . The tail end of the polyethylene tube was connected to a 50-Before the topical application of each solution, blood flow was measured twice at 30-min intervals. When the blood flow had stabilised, the solution was dropped on the region of measurement, and 30 min later the blood flow was measured again. The rate of change of blood flow (%) was calculated from the values before and 30 min after the topical application of the solution.μl of 0.2 mg ml−1 AC7700 solution on the vessels of the s.c. tissue, and the measurements were recorded.For direct observation of the contraction of normal arterioles caused by AC7700, intravital microscopy was used to drop 10 6) were implanted in the right kidney as described earlier. At 7–10 days after the implantation, the second incision was made at exactly the same incision site as that of the first incision, the kidney was exposed, and hydrogen electrodes were inserted into the tumour growing there. Next, a polyethylene tube with an outer diameter of 0.61 mm  was inserted into the left common carotid artery until the tip of the tube reached a position 2–3 mm upstream of the right renal artery. All incision wounds were sutured with thread or were closed with synthetic resin glue (Aron Alpha 201). Certain animals in the i.v. injection group also underwent this operation as a sham operation.For this comparison, LY80 cells (2 × 10n=6) and the i.v. injection group (n=6), 10 mg ml−1 AC7700 was infused at a rate of 0.15 ml min−1 using a microinfusion pump (Harvard Apparatus Co.). The final dose administered was 10 mg kg−1. After administration of AC7700, TBF was measured at multiple time points  for 6 h. In another experimental group, AC7700 was administered i.a. to rats at a dose of 0.5 and 1 mg kg−1.In both the i.a. injection group  feeding arterioles, which supply blood to a tumour vascular network; (b) tumour capillaries, which constitute most of a tumour vascular network and play an important role in nutrient exchange; and (c) drainage vessels, which drain blood from a tumour vascular network. We previously reported that TBF is controlled by modified a2 vessels (−1) was administered via the lateral tail vein using an infusion pump (Harvard Apparatus Co.). The changes in TBF and the tumour vascular network were directly observed via a light microscope  with a × 10 ocular  and × 2-20 objectives . Tumour vessels within the chamber were transilluminated by a 12-V 100-W halogen lamp. The microscopic image was recorded using a closed-circuit video system consisting of a CCD video camera , a TV monitor , and an S-VHS video recorder .After anaesthesia, a rat with a transparent chamber was placed in the right lateral position on a heated stage  at 34.5°C, which was attached to the mechanical stage of the microscope. AC7700  was injected into the rat as a single i.v. bolus. Tumour vessels within the chamber were epi-illuminated through a primary filter (420–490 nm), a 505-nm dichroic interference mirror, and a 520-nm barrier filter. The fluorescein microscopic images were photographed using a silicone-intensified video camera .A video timer was superimposed on the images for record keeping. Segments of the videotape containing desired images were transferred to the hard disk of a computer  through a video frame grabber board . Final images were output by a digital printer .Intravital microscopic observation for diameter measurements was performed under × 200 magnification. Changes in arteriole and drainage diameters caused by AC7700 were measured directly on the TV monitor. The rate of change in the diameter before and after the administration of AC7700 was calculated.−1 AC7700 was administered. FITC-dextran was then injected 30 min after the AC7700 administration to demonstrate complete loss of circulatory function in the tumour vascular network. After an additional 90 min, the glass window of one side of a transparent chamber was gently removed from the rat, 15% formalin solution was put directly on the tissue (approximately 150 μm thick), and the thin tissue was fixed before killing. The tissue was processed and embedded in paraffin. Sections (4 μm thick) were cut in parallel to the surface of the membrane tissue within the transparent chamber and were stained with haematoxylin and eosin. Changes in microvessels caused by AC7700, in particular, changes in tumour vascular lumens, were carefully assessed.To confirm the presence of functioning tumour vessels, 2% FITC-dextran was injected i.v. with the use of intravital microscopy. When FITC-dextran disappeared from the tumour tissue 120 min later, 10 mg kgThe tumour interstitial fluid pressure was measured by two different methods – the diffusion chamber method and the wick-in-needle method. Systemic arterial pressure and TBF were also simultaneously measured in both methods.μm, developed originally by 6 LY80 tumour cells was injected s.c. around it. The chamber was completely enveloped in the LY80 tumour approximately 1 week after transplantation of tumour cells. The TIFP was recorded electrically with a transducer  through the polyethylene tube connected to the diffusion chamber. The tumour interstitial fluid pressure was serially measured and recorded before and after AC7700 administration.We previously reported this method in detail  solution was dropped directly at the site of blood flow measurement, the flow signal increased in a transient manner as an artefact; however, the blood flow returned to its original value within 5 min. Each blood flow response caused by topical application of each concentration of AC7700 was reproducible.Blood flow values measured by the special electrodes developed for topical application were not significantly different from those measured by electrodes that have been used so far (data not shown), which indicated that the slight increase in the diameter of the electrodes because of the covering wire did not greatly influence the blood flow measurement. When 10 μl of various concentrations  of the AC7700 solution or 10 μl of 0.9% NaCl solution was dropped directly at the site of blood flow measurement in normal s.c. tissues and s.c. tumours. Local blood flow of the normal s.c. tissues decreased significantly and dose dependently because of AC7700 . In contrast, local blood flow of LY80 tumours did not change significantly at any concentration of AC7700 . The TBF did decrease significantly with 50 mg ml−1 AC7700 (P=0.0002).Figure 1A−1 in tumour tissue, whereas only 1 mg ml−1 was sufficient in normal tissue. That is, the sensitivity of the blood flow response after topical application of AC7700 was markedly higher in normal vessels relative to the tumour vessels. Even at the concentration of 0.2 mg ml−1, when AC7700 was applied directly to normal s.c. tissue, arterioles first constricted . Intra-arterial administration of AC7700 did not have stronger effects than i.v. administration. For a dose of 1 mg kg−1 AC7700 or lower, i.a. administration did not induce a prominent decrease in TBF (data not shown).A comparison of changes in TBF after i.v. and i.a. administration of 10 mg kg−1 AC7700 was given i.v., all orders of arterioles within a transparent chamber showed significant vasoconstriction . The changes in diameters of a4, a3, and a2 vessels 30 min after AC7700 administration were −29.2, −33.6, and −47.2%, respectively. The blood flow in tumour-feeding vessels (modified a2 arterioles) ceased completely, and the vessels disappeared from view. Thus, it was impossible to measure the diameter of these vessels after AC7700 administration. The blood flow in normal tissue did not stop.A typical intravital microscopic finding showing the process of change in tumour microcirculation caused by AC7700 is shown in Figure 3stricted , and thestricted  began toμm. However, 30 min after AC7700 administration FITC-dextran did not reach the tumour vessels at all (data not shown) because of complete blockage of the tumour microcirculation. A histological section of the same area of the tumour tissue, which was locally fixed by 15% formalin before killing, is shown in When FITC-dextran was administered by a single bolus i.v. injection before AC7700 administration, the fluorescent dye reached the tumour vessels  caused by 10 mg kg−1 AC7700. The mean tumour size, MABP, TIFP, and TBF at the start of measurement were 3.5±0.4 cm3, 96.4±7.1 mmHg, 10.5±1.6 mmHg, and 23.5±16.6 ml min−1(100 g)−1, respectively. When AC7700 was administered i.v., MABP increased, and TBF and TIFP simultaneously decreased. The MABP, TIFP, and TBF 30 min after AC7700 administration were 120.0±4.0 mmHg, 4.8±0.5 mmHg, and 2.5±1.7 ml min−1 (100 g)−1, respectively. These parameters did not change at all after i.v. administration of 0.9% NaCl solution (data not shown). Changes in MABP, TIFP, and TBF induced by AC7700 were all significant .When a diffusion chamber was implanted in the s.c. tissue of the back of a rat and a tumour cell suspension was injected around the chamber, the chamber was completely enveloped in the growing tumour 1 week later. Figure 73 (n=6). After i.v. administration of 10 mg kg−1 AC7700, MABP significantly increased from 95.2±7.8 (before the drug) to 137.3±10.7 mmHg (30 min after administration) (P<0.0001), TIFP significantly decreased from 12.1±3.1 (before) to 7.1±2.3 mmHg (30 min after) (P=0.001), and TBF significantly decreased from 30.2±6.1 (before) to 5.5±1.9 ml min−1 (100 g)−1 (30 min after) (P<0.0001). The results obtained by this method supported those of the diffusion chamber method.At the start of measurement using the wick-in-needle method, the mean tumour size was 6.4±3.0 cmWe conclude that TBF stasis brought about by AC7700 administration most likely results from an indirect action involving the contractile response of arterioles, rather than a direct effect of this drug on tumour vessels. Our experiments in the present study strongly support this conclusion.−1. In normal tissue, however, there was strong constriction of the arterioles with 1 mg ml−1 AC7700, as well as a large decrease in blood flow in the capillary bed. We therefore conclude that the small changes in TBF were not due to an insufficient volume of AC7700 having been applied to the region, but instead were due to the relatively low sensitivity of the tumour vessels themselves. Thus, it was strongly suggested that the stasis of TBF induced by AC7700 administration was not caused by a direct effect of the drug on the tumour vasculature.In the first experiment, the sensitivity of vessels in tumours to topical application of AC7700 was compared with that of vessels in normal s.c. tissue. Normal tissue was more sensitive than tumour tissue to this agent. When blood flow volume was measured via the hydrogen clearance method, what was actually measured was not the total blood flow , but theIn the second experiment, the effects of i.v. and i.a. administration of AC7700 were compared. If AC7700 works directly on tumour vessels, then the stasis effect after arterial administration – leading to a high concentration of AC7700 reaching the tumour – should be stronger than that after venous administration. In fact, stronger stasis was not obtained with i.a. administration. This finding strongly suggests that the site of AC7700 action is not the luminal surface of tumour vessels.The results of the above experiments indicate that the primary action of AC7700 is not on the tumour vasculature. If that is the case, then why indeed does AC7700 bring about TBF stasis and why does not the stopped TBF subsequently recover? To answer these questions, we used a rat transparent chamber to observe the effects of AC7700 on host arterioles and the tumour vascular network.We previously reported that the fact that the sites of increased vascular resistance differ with each vasopressor is the primary reason why various vasopressors produce different changes in TBF , but if haemolysis occurs in the drainage region the TBF stasis becomes irreversible. Since haemolysis acts as a vascular toxin and is likely to be a cause of vascular destruction, it is thought to be the direct cause of irreversible TBF stasis. The precise reason for the haemolysis remains uncertain, and additional studies of this phenomenon are needed.in vivo experiments that CA-4-P increased tumour vascular permeability. Moreover, in the process of increasing permeability, CA-4-P bound to tubulin and was surmised to be involved in causing the cytoskeletal disorganisation of the endothelium (4An rat gliomas, CA-4-P did not produce an increase in TIFP. These conflicting findings suggest that there may be differences in tumour vascular permeability and TIFP brought about by CA-4-P that are related to the type of tumour.Recently, The finding that CA-4-P does not immediately constrict the arteriolar system whereas AC7700 immediately constricts it suggests that their similar chemical structures result in similar effects via different responses in the vascular system. That the pharmacological effects of these two similar drugs differ, or that sometimes the drugs produce opposite effects, is not unusual in pharmacology. For example, it is well known that both epinephrine and isoproterenol are catecholamines with similar structures, but they produce diametrically opposed effects on peripheral vascular resistance. It would therefore not be surprising if AC7700 and CA-4-P also have different mechanisms of action.In light of the experimental findings obtained in the present study, we can summarise the effects of AC7700 as producing TBF stasis that, in turn, brings about necrosis of solid tumours, as shown in Figure 8


Over the past 70 years many experiments have been designed to promote tumour growth. These studies were all carried out in allogeneic tumour systems or by artificially influencing the immunization process. In the present study, the growth of syngeneic mammary tumour cells was enhanced by prior immunization via the intracaecal route. Such induced enhancement could be transferred to untreated animals by serum or by spleen cells. Tumour growth was also enhanced in another syngeneic system by immunization via the intestinal route with frozen-thawed ascites tumour cells. The result is in direct contrast to that obtained by similar immunization with live cells, which affords protection against a later challenge.


Lymphocytes from 10 patients with breast carcinoma were seeded in autologous serum, on autochthonous tumour cells and allogeneic tissue-cultured breast tumour cell lines. In 4 patients, the anti-tumour cell cytotoxicity against at least one of 3 breast tumour cell lines differed significantly from that against autochthonous tumour cells. Further study of these 4 individuals (using their previously frozen lymphoid cells and sera) showed that these differences occurred because serum which decreased ("blocked") lymphocyte anti-tumour cytotoxicity when applied to one tumour cell line, could either have no effect or potentiate it when applied to another, without any consistent pattern vis-à-vis target-cell susceptibility to these different humoral effects.


B) receptor agonist IRL 1620 were investigated in the rat P22 carcinosarcoma and a range of normal tissues in BDIX rats. Tissue blood flow rate was calculated from measurements of tissue uptake of radiolabelled iodoantipyrine. A comparison of vascular effects in the P22 tumour and the HSN sarcoma growing in CBH/CBi rats was made using laser Doppler flowmetry, showing similar effects of IRL 1620, with red cell flux rapidly decreasing by 50–60% and then returning to control levels within approximately 30 min. This corresponded to similar levels but different spatial organisation of ETB binding sites in the two tumours, as measured by autoradiography. The decrease in tumour blood flow and an increase in vascular resistance suggest that the vascular component of ETB receptors in the P22 tumour is localised on contractile elements rather than on endothelial cells. ETA receptors were also identified. Vasoconstriction occurred uniformly throughout the P22 tumour mass, consistent with a measured homogeneous distribution of ETB receptors. IRL 1620 caused vasoconstriction in normal skeletal muscle, kidney and small intestine of the BDIX rat as well as in tumour, but did not affect blood flow in other tissues. These effects could be useful for limiting toxicity of certain chemotherapeutic agents. Fully functional ETB receptors are clearly expressed on tumour vasculature and IRL 1620 shows promise for short-term modification of tumour blood flow. Expression levels of ETB receptors on the tumour vasculature could be useful for predicting which tumours are likely to respond to IRL 1620.The vascular effects of the endothelin B (ET Tumour vasculature is morphologically and functionally abnormal . TherefoEndothelin-1 (ET-1), together with ET-2, ET-3 and the ET receptors , exerts many important physiological roles in normal tissues, as well as in cancer (A) and endothelin B (ETB) receptors. ETA receptors are mainly found on vascular smooth muscle cells and pericytes and these account for the profound vasoconstriction of ET-1. ETB receptors are found on contractile elements and endothelial cells and their activation can cause both vasoconstriction (activation of receptors on vascular smooth muscle cells/pericytes) and vasodilatation  (The vasoactive action of ET-1 is mediated primarily via endothelin A (ETl cells) .B receptor agonist IRL 1620. Surprisingly, the HSN tumour vasculature constricted relatively selectively in response to this agonist compared with the majority of normal tissues, with the exception of the small intestine  and investigating a full time course of vascular response in tumours Early generations of the P22 transplanted rat carcinosarcoma were grown subcutaneously in the left flank of 10- to 12-week-old male BDIX rats as described previously . Tumours9, Ala11,15)-ET-1(8–21)]),  was dissolved in physiological saline and administered to the rats by slow bolus intravenous (i.v.) injection, at a dose of 3 nmol kg−1 in an injection volume of 4 ml kg−1, which was an effective dose in the HSN tumour model (IRL 1620 ([suc-(Glu−1), fentanyl citrate (0.315 mg kg−1)  and midazolam (2 mg kg−1)  and kept warm using a thermostatically controlled heating pad. Five microprobes were randomly inserted into each tumour through skin incisions, to a depth of at least 1 mm. After a 20 min stabilisation period, animals were treated with IRL 1620 and RBC flux was monitored continuously for 70 min. Measurements were continued for at least a further 5 min following a lethal injection of anaesthetic to allow the biological zero RBC flux to be measured. From the 20 readings per second recorded, 3 min averages were calculated for each probe. After the subtraction of biological zero, the data were converted to the relative RBC flux, expressed as the percentage of the RBC flux at time 0 (time of injection of IRL 1620). Mean values for each animal at each time point were calculated from the individual probe values and these were used to calculate the overall mean value for relative RBC flux, at each time point, for the whole treatment group.Red blood cells (RBC) flux was monitored using the Oxford Array multiple channel laser Doppler system . Rats were anaesthetised by intraperitoneal (i.p.) injection of fluanisone (10 mg kg14C-labelled iodoantipyrine (14C-IAP)  and 125I-labelled iodoantipyrine (125I-IAP)  has been described previously  was administered to the rat. The rats were heparinised 5 min prior to blood flow measurement via a venous catheter ). Free flowing arterial blood was collected into preweighed vials at 1 s intervals, during a 30 s i.v. infusion of 0.56 MBq (15 μCi) 14C-IAP or 0.37 MBq (10 μCi) 125I-IAP prepared in 0.9% saline. Infusion was achieved via a constant flow infusion pump set at a rate of 1.6 ml min−1, which compensated for the blood loss from the arterial circulation and maintained blood pressure constant (unpublished data). At 30 s, the rat was rapidly killed by i.v. injection of Euthatal (0.3 ml)  and the tumour and selected normal tissues were rapidly excised. When using 125I-IAP, blood and tissues were weighed and the 125I levels determined using a Wallac  Autogamma well counter. In the case of 14C-IAP blood flow measurements, no normal tissues were excised. Blood samples were weighed and 10 μl aliquots were placed in liquid scintillation vials to which 1 ml Soluene  was added. Vials were left overnight, and then 10 ml scintillation fluid  was added. Radioactivity was determined by liquid scintillation counting  with suitable quench correction. Immediately after excision, the tumours were mounted in Tissue-Tek embedding compound , frozen in isopentane at −40°C and stored at −70°C for subsequent processing. Cryostat sections (20 μm thick) were cut from each tumour, mounted onto poly-L-lysine-coated slides  and rapidly dried on a hot plate. Slides were overlaid with autoradiographic film  for up to 2 weeks, alongside 14C-labelled methylmethacrylate microscales  of known isotope concentration for quantification of autoradiograms. Following development of autoradiograms, sections were fixed in methanol and stained with haematoxylin and eosin (H&E).At different time points  before the determination of blood flow, a 3.0 nmol kg−1 min−1 from the tissue counts measured via either autoradiography or gamma counting, the equilibrium partition coefficient of IAP in the different tissues and the arterial input function derived from the arterial blood counts. The mathematical model used was adapted from Blood flow to the tumour and normal tissues was calculated in ml gA single blood flow value was obtained for each tissue, where counts were obtained by gamma counting. Autoradiographic images were captured by camera and transformed pixel by pixel into blood flow rate images using the same principles as described above. Analysis was performed using in-house software developed using the Visilog Image Processing package . Blood flow autoradiogram images were overlaid onto histological images from H&E-stained sections to exclude necrotic regions from the calculations of blood flow. Tumour peripheral regions were defined as the rim of the tumour equal to 10% of the section diameter, the remaining portion of the section being defined as the central region.A and ETB receptors in untreated P22 tumours was assessed on sections of the same tumours as used for the blood flow determination. Preliminary experiments were carried out to determine suitable incubation concentrations of compounds and suitable incubation and washing times to maximise specific binding (data not shown). Slides containing serial pairs of tumour sections were incubated in wash buffer   to remove endogenous peptides, drug and 14C-IAP. Sections were dried at room temperature in air, then incubated for 90 min with 125I-ET-1 (0.13 nM) plus phosphoramidon  (a) alone , or with the addition of either (b) BQ-788 , or (c) BQ-610 . The further inclusion of cold ET-1 (1 μM) to each group allowed the determination of nonspecific binding in each case. After incubation, sections were rinsed three times in wash buffer, dried in air and exposed to Hyperfilm-3H (Amersham) for 24 h alongside 125I-microscales. Hyperfilm-3H has no scratch-resistant coating and is therefore suitable for detection of Auger electrons, which are emitted in addition to gamma-rays during the decay of 125I. Exposure of test sections for varying times showed that 24 h was the optimum time for obtaining grey levels that covered the dynamic range of the film. Autoradiograms were developed and sections fixed in methanol and stained with haematoxylin and eosin.Distribution of ET125I-microscales were converted to total and nonspecific binding (fmol mg−1 protein) using the specific activity of the 125I-microscales supplied by the manufacturer (Amersham). These modified scales were used to generate standard curves from which optical density of the images of the tumour sections were transformed into total, specific and nonspecific binding images. Specific binding for each pair of sections used for receptor studies was calculated by subtraction of the mean nonspecific binding from the mean total binding using the whole sections as the region of interest. Image analysis was carried out using in-house software developed using the Visilog Image Processing package.Levels of radioactivity in the P-values less than 0.05 were taken as indicative of significant differences between groups. Data are presented as arithmetic means±s.e. (standard error of the mean). Temporal blood flow changes for the radiotracer data were analysed using linear regression and the computed curves were compared for significance using ‘overall test for coincidence of the two regression lines’  followed by a Tukey–Kramer post-test; n lines’ . Laser D−1 decreased RBC flux immediately after injection, as measured by Laser Doppler flowmetry in both tumour models  and ETA binding tended to be higher in the tumour periphery compared to tumour centre, but this was not statistically significant  and ETA receptor binding with 78 and 85% specific binding, respectively, compared with 51% specific binding for ETB receptors.The distribution of total , suggesting that vasoconstriction in kidney is mediated primarily through the ETB receptor. This was also suggested by A receptors .To assess the regional changes in tumour blood flow after treatment with IRL 1620, we used tumour uptake of A, as well as ETB receptors, with binding sites distributed relatively homogeneously across the tumour section. A high degree of specific binding, in the region of 85% of the total binding, was observed when ETA+ETB receptors were observed together and when ETA receptor binding was performed (in the presence of the ETB antagonist BQ788). In contrast, a much lower intensity of ETB receptors was observed in P22 tumour sections (in the presence of the ETA antagonist BQ610), which corresponded to a lower level of specific binding (51%). Previous workers have reported the binding kinetics of ET-1 at ETA and ETB receptors, and have observed the dissociation constants (Kd) of the ligand to be similar at each receptor (A receptors than ETB receptors in the P22 tumour, rather than merely reflecting differences in the selectivity and affinity of the ligand for each receptor subtype.Receptor binding studies on cryostat sections of the same tumours as used in the blood flow analysis showed that the P22 tumour possesses ETB receptor binding in the rat HSN tumour was concentrated in small regions of intense binding (B receptor distribution in P22 tumours was homogeneous and suggests that ETB receptors are localised on tumour cells as well as on the vasculature in this tumour type. Similarly, in human glioblastoma tumours, the pattern of ETB receptor distribution was homogeneous suggesting localisation on cancer cells, whereas ETA receptors were highly expressed in glioblastoma vessels and in some scattered glioblastoma areas (B receptor binding site distribution, vascular resistance in HSN and P22 tumours was increased to approximately the same degree. Over the whole tumour sections, specific ETB receptor binding in the P22 tumour was 1.7 fmol mg−1 (−1 for the HSN tumour (ETmol mg−1  comparedA antagonists (B agonist, IRL 1620, would be useful for short-term blood flow reduction in tumours expressing the ETB receptor and, due to its constrictive effect, IRL 1620 may also have antiangiogenic properties. It would be important to determine the effects of this ET-1 agonist in tumours that endogenously produce high levels of ET-1 such as prostate, breast and ovarian cancers (In most human tumours investigated so far, both ET receptor subtypes have been identified (B receptors are expressed on the vasculature of at least two different tumours, leading to a relatively selective and profound short-term reduction of tumour blood flow, following administration of the ETB receptor agonist, IRL 1620. Expression levels of ETB receptors on the tumour vasculature could be useful for predicting which tumours are likely to respond to IRL 1620. The vasoconstriction produced in the P22 and HSN tumours was equivalent to or greater than that found for normal tissues and to our knowledge, this is a unique feature of IRL 1620 compared to other vasoconstrictors, which tend to be less effective against tumour than normal blood vessels. The activation of ETB receptors with selective agonists such as IRL 1620 is therefore promising, with the proviso that normal kidney, intestine and skeletal muscle are also affected. Distribution of ETB binding in the P22 tumour suggested ETB receptor localisation on tumour cells as well as on the vasculature, which may also be significant for tumour growth. Blood flow results suggested that the vascular component of ETB receptors is localised on contractile elements rather than on endothelial cells. Considering the profound vasoconstrictive effect of a single dose of IRL 1620 in tumours, further studies are warranted to determine its effects in a repeated dosing schedule on tumour angiogenesis and growth, where selectivity of vascular effect for tumours may not be critical. In addition, further studies using specific ET-1 receptor agonists and antagonists should provide more insight into the role of the endothelin axis in the tumour vasculature, leading to the development of specific vascular-targeted therapies.In conclusion, our results show that fully functional ET


Human alpha fetoprotein (AFP) has been detected by the agar double diffusion method in ascitic fluid, cerebrospinal fluid (CSF) and bile, from fetuses, neonates and patients with AFP seropositive hepatocellular carcinoma. AFP was detected in the meconium and faeces of fetuses and neonates respectively. The protein was not detected in the amniotic fluid nor the pericardial fluid. It was found in the urine in only two fetuses that had concomittant renal disease. It was not detected in breast milk of lactating females. When metastases occurred in the lung from a hepatocellular carcinoma producing AFP, the pleural effusions sometimes contained AFP. The concentrations of AFP in the serum and in the other body fluids were about the same. This indicates that other body fluids can be used for the diagnosis of hepatocellular carcinoma.


We analysed the effect of graft-contaminating tumour cells on the long-term survival of 24 patients with high-risk neuroblastoma and found that patients whose grafts contained detectable neuroblastoma cells had a significantly higher probability of survival than did patients with no detectable tumour cells. Estimated contamination of the graft by more than 2000 tumour cells was associated with a significantly higher probability of survival than contamination with fewer tumour cells. We hypothesise that the presence of a critical number of graft-contaminating neuroblastoma cells can elicit a protective antitumour immune response after autologous transplantation. Induction of a tumour-specific immune response is a major goal in the treatment of patients with solid tumours , and proA clinical scenario that most closely mimics those described in mice is autologous transplantation with a tumour-contaminated stem cell graft during chemotherapy-induced neutropenia and lymphopenia. Such a scenario is often encountered in children with high-risk neuroblastoma, up to 82% of whom have tumour contamination of their peripheral stem cell grafts  were included into the retrospective analysis. In all patients, peripheral blood stem cells (PBSC) were mobilised and collected as described  and the median to reach 0.5 × 109 l−1 neutrophiles after myeloablative therapy was 12 days (range 8-24 days). Lymphoid reconstitution was analysed in eight patients and the median to reach >0.2 × 109 and >1 × 109 l−1 CD3+ T lymphocytes was 21 days and 6 months, respectively. Before purging, 17 of 22 grafts tested contained various numbers of tumour cells (0.82 × 106–5.2 × 106 tumour cells per graft). Tumour contamination was evaluated in all 24 grafts after purging. Four of the 24 grafts contained measurable neuroblastoma cells  and all four patients are long-term survivors after autografting. In contrast, the probability of 5-year survival among the other 20 patients was 33±11%   the possible absolute number of neuroblastoma cells in the 20 grafts that had no measurable tumour cells. We assumed maximal contamination based on our observation of tumour-cell contamination in 17 of the 22 evaluable unpurged grafts and the fact that more sensitive methods of tumour detection, such as polymerase chain reaction, often reveal the presence of tumour cells after purging (n=10) had a significantly larger number of tumour cells in their grafts than did patients who died of relapse (n=14)  but only 19±10% for those estimated to be infused with fewer than 2000 tumour cells (n=16) . The number of transplanted CD34+ stem cells or the remission status at the time of autologous transplantation had no influence on the outcome (data not shown).In order to estimate whether there would be a threshold number of protective graft-contaminating tumour cells, we calculated on the basis of the lower detection limit of our assay , which are mostly tissue-specific or differentiation antigens encoded by normal genes , are tar+ cell selection might also play a role in survival by preventing the infusion of higher numbers of tumour cells that might overwhelm the immune system and result in relapse (Our observations suggest that graft-contaminating neuroblastoma cells induced a tumour-protective immune response after transplantation. If so, the initiation of such a response may have required a threshold quantity of tumour antigen. This would explain the association between long-term survival and the presence of more than 2000 estimated tumour cells in the graft. The reduction of the number of contaminating tumour cells by CD34


In addition core biopsies were obtained from the same tumour on two separate occasions (n = 10). A highly significant correlation was found in counts performed by the same observers at different times and between two different observers. No significant correlation was found in counts of core biopsies and tumour sections taken either simultaneously or subsequently. No correlation was found between counts of sequential core biopsies. Study findings suggest that, although microvessel counts may be assessed reproducibly by the same and different observers, counts performed in core biopsies do not accurately reflect those of overall tumour, limiting their potential as predictive or prognostic markers. © 1999 Cancer Research CampaignAssessment of tumour vascularity in core biopsy specimens may be a useful predictor of response to primary therapy. This study addresses practical methodological issues regarding accuracy of tumour vascularity assessments in different breast cancer specimens. Issues addressed in the study are variation caused by (i) inherent observer variation in the method, (ii) tumour heterogeneity and (iii) previous surgical manipulation of tumours. Microvessel counts were performed by two observers on separate occasions and by two different observers. Counts were performed on core biopsies and tumour sections taken simultaneously (


The synthesis and degradation of type I and type III interstitial collagens releases several antigenic metabolites, whose measurement allows the metabolism of connective tissue to be evaluated under a variety of different conditions. In this study we investigated the influence of benign and malignant ovarian neoplasms on the metabolism of these collagens. The study population comprised patients with benign (n = 53), borderline (n = 6) or malignant (n = 36) ovarian neoplasms. We quantified the serum, cyst fluid and peritoneal/ascitic fluid concentrations of the amino-terminal propeptide of type I (PINP) and III (PIIINP) procollagens, indicators of the synthesis of type I and III collagen, respectively and the cross-linked carboxy-terminal telopeptide of type I collagen (ICTP), an indicator of type I collagen degradation. Macrophage colony-stimulating factor 1 (CSF-1) concentration was also assayed as its serum level is increased in ovarian cancer and CSF-1 may be involved in the regulation of collagen metabolism. The concentration of each antigen was significantly higher in patients with malignant tumour than with benign neoplasm in each comparison, except for ICTP in peritoneal fluid and for CSF-1 in cyst fluid. The high ascitic fluid concentration of PINP, PIIINP or CSF-1 correlated with malignancy, and the low cyst fluid concentration of any of the four markers was indicative of benign tumour. Levels of CSF-1 did not correlate with the levels of any of the markers of collagen turnover. The concentration of PINP in ascites was about 50 times higher and in cyst fluid about eight times higher than that in the serum from patients with malignant tumour, whereas the respective ratios for ICTP were only 2.5 and 1.3. In such patients, the ratio of ascitic fluid to serum concentration was also about 80-fold higher for PIIINP and about 20-fold higher for PINP than for ICTP. The different distributions of PIIINP, PINP and ICTP suggests dominance of synthetic processes or retarded elimination of PIIINP and PINP in ovarian cancer. In advanced malignancies, the accumulation of PINP and PIIINP in abdominal space, possibly due to increased synthesis and/or failed resorption, may promote ascites formation. This study shows that both accelerated synthesis and breakdown of fibrillar collagens are characteristic of ovarian malignancy, and suggests that measurements of cyst fluid or ascitic fluid concentrations of collagen metabolites or CSF-1 could be used in the differential diagnosis of benign and malignant ovarian neoplasms.


The assessment of a tumour's angiogenic potential, by measuring the microvessel density in histological sections, assumes that a 4-microm section is representative of whole tumour vascularity. This study has examined this assumption by comparing the vessel density found radiologically, after injecting specimens with contrast, with that found immunohistochemically. Twenty-one breast angiograms were performed following mastectomy for carcinoma and graded 1-3 for vessel density. Sections (4 microm) from these carcinomas were labelled for endothelial cells using anti-CD34, and the vessel counts were compared with the radiological grades. A significant correlation was found between the densities . We therefore conclude that the microvessel density measured in histological sections is representative of whole tumour vascularity.