Suppressive immune cells present in tumour microenvironments are known to augment tumour growth and hamper efficacy of antitumour therapies. The amino-bisphosphonate Zoledronic acid (ZA) is considered as an antitumour agent, as recent studies showed that ZA prolongs disease-free survival in cancer patients. The exact mechanism is a topic of debate; it has been suggested that ZA targets tumour-associated macrophages (TAMs).in vivo and in vitro. Mice were intraperitoneally inoculated with a lethal dose of mesothelioma tumour cells and treated with ZA to determine the effects on myeloid differentiation and survival.We investigate the role of ZA on the myeloid differentiation to TAMs in murine mesothelioma We show that ZA impaired myeloid differentiation. Inhibition of myeloid differentiation led to a reduction in TAMs, but the number of immature myeloid cells with myeloid-derived suppressor cell (MDSC) characteristics was increased. In addition, ZA affects the phenotype of macrophages leading to reduced level of TAM-associated cytokines in the tumour microenvironment. No improvement of survival was observed.We conclude that ZA leads to a reduction in macrophages and impairs polarisation towards an M2 phenotype, but this was associated with an increase in the number of immature myeloid cells, which might diminish the effects of ZA on survival. Recently, cancer-related inflammation was recognised as the seventh hallmark of cancer .highGr-1low-MDSC , whereas classically activated macrophages (M1) are more associated with inflammation. These two types of macrophages differ in receptor expression, effector function, and cytokine and chemokine productions .The recruitment of immature myeloid cells is enhanced by the production of chemokine (C–C motif) ligand-2/monocyte-chemotactic protein-1 (CCL-2/MCP-1), which is produced and expressed by TAMs and tumour cells. Within the heterogeneous group of immature myeloid cells, subpopulations are defined, which can further differentiate into mature macrophages, and hence TAMs provide their own precursor cells, which enhances tumour progression  , on TAM formation and tumour progression in a murine mesothelioma model.in vitro and in vivo. In addition, we determined whether ZA changed the phenotype of macrophages. Animal studies were performed to investigate the effect of ZA on survival.In this study, we analysed the effect of ZA on myeloid differentiation both CBA-j mice  were purchased from Harlan  and were housed under pathogen-free conditions at the Erasmus MC animal facility. All experiments were approved by the local ethical committee for animal welfare  and complied with the guidelines for the welfare of animals in experimental neoplasia by the United Kingdom Coordinating Committee on Cancer Research (UKCCCR) and by the Code of Practice of the Dutch Veterinarian Inspection. The AC29 cell line is kindly provided by Professor BWS Robinson .Macrophages were depleted by intraperitoneal (i.p.) injection of liposome-encapsulated clodronate  .6 AC29 tumour cells on day 0. Mice were i.p. injected with 200 μl liposome-encapsulated clodronate or liposome-encapsulated PBS at days 5 and 10 after tumour injection. At day 12 mice were killed using CO2. The peritoneal cavity of tumour-bearing mice was washed with 1 ml PBS to obtain peritoneal cells for FACS analysis. All visible tumour material was excised from each mouse and data are expressed as wet weight (accuracy of 0.001 g).CBA-j mice were i.p. inoculated with a lethal dose of 20 × 10Tumour-associated macrophages were defined by the expression of F4/80, MHCII and CD206 (marker for M2 macrophage phenotype).μm) were cut in an HM-560 cryostat . F4/80  and CD206  primary antibodies were incubated for 1 h at room temperature. Binding of antibodies was detected using the immunoalkaline phosphatase antialkaline phosphatase method . Naphthol-AS-MX-phosphate  and new fuchsine  were used as substrate. The specificity of the antibodies was checked using a protein concentration-matched non-relevant monoclonal antibody and PBS.Tumour material was obtained 25 days after tumour injection from the peritoneal cavity of tumour-bearing mice. Tumour biopsies were embedded in Tissue-Tek II optimum cutting temperature medium , snap frozen in liquid nitrogen and stored at −80°C. Tissue sections , depleted of erythrocytes by osmotic shock, washed twice in RPMI and adjusted to a concentration of 1 × 106 cells ml−1 in FACS buffer.Spleens were aseptically removed and mechanically dispersed in cold PBS. Cell suspensions were filtered through a 100-Splenocytes were stained with the following monoclonal antibodies: Ly6C (FITC), F4/80 (FITC), MHCII (PE), CD11c (PE-Texas red), CD11b (PerCP-Cy5.5), CD31 (PE-Cy7), CD206 (Alexa 647), Ly6G (Alexa Fluor 700), Gr-1 (APC-Cy7) and a fixable live/dead marker in DAPI . The final analysis and graphical output were performed using FlowJo software .−1)  and β-mercaptoethanol (Sigma-Alderich) (further referred to as culture medium).Single-cell suspensions of bone marrow, isolated from femurs and tibias of naïve mice, were cultured in RPMI supplemented with 10% foetal calf serum, 2.5 ml gentamicin  was used in our bone marrow cultures according to the protocol of 6 bone marrow-derived cells. AC29 was cultured in RPMI-1640  supplemented with GlutaMax, 10 mM HEPES and 5% heat-inactivated foetal bovine serum. When confluency reached 80% , supernatant was collected, centrifuged for 10 min at 1000 g to remove cells/cell debris. Supernatant is added to the bone marrow culture at a concentration of 30%. This concentration was selected based on findings by μM. All culture experiments have been repeated five times under comparable conditions.Bone marrow cells were isolated from the femurs and tibias of naïve mice under sterile conditions (Mononuclear-MDSCs were isolated from the spleen of tumour-bearing mice. Four to six colour samples were sorted using a FACSAria equipped with FACSDIVA software (BD Biosciences). After sorting, cells were cultured with M-CSF for 6 days. This experiment has been repeated twice under comparable conditions.6 AC29 tumour cells. One group was treated daily with subcutaneous (s.c.) ZA injections  in 100 μl PBS and the other group was treated with 100 μl PBS as a control starting at day 5. This dosage was proven effective and non-toxic in mice  and VEGF levels in the effusion fluid were determined using a specific ELISA assay : BD Biosciences; VEGF: Calbiochem, Darmstadt, Germany). Protocols were followed as per the manufacturer's instructions. Samples were diluted appropriately to ensure that readings were within the limits of accurate detection. Results are expressed as pg ml−1 of effusion fluid.To measure cytokine levels, effusion fluid from the peritoneal cavity of tumour-bearing mice was obtained at day 25 after tumour injection. IL-10, IL-12, TNFt-tests. A two-tailed P-value <0.05 was considered significant. Data presented as a percentage of tumour-free animals were analysed with Kaplan–Meier survival curves, using the log-rank test to determine significance.Data are expressed as mean±s.d. Comparisons between groups were made using We investigated the effect of the depletion of macrophages on tumour progression in a murine model for mesothelioma by treating mice with liposome-encapsulated clodronate. These liposomes are readily taken up by phagocytic cells, including macrophages, and induce cell-specific apoptosis after clodronate is set free into the cytoplasm of cells .Mice were i.p. inoculated with a lethal dose of AC29 tumour cells and mice were treated with liposome-encapsulated clodronate or liposome-encapsulated PBS on days 5 and 10 after tumour injection. Mice were killed 12 days after tumour injection. All visible tumour material was excised from each mouse and wet weight was measured . All mice (n=5) treated with liposome-encapsulated PBS showed profound tumour growth at day 12. Three of the five mice treated with liposome-encapsulated clodronate had no visible tumour. In the case of mice that did develop tumour, tumour growth was less profound . The expression of Gr-1 remained high in both these culture conditions  of these markers on day 6 of culture (nditions .P<0.0001)  .in vitro, leading to a significant reduction in the MFI of CD206 on macrophages cultured in the presence of ZA (P<0.0001).In conclusion, these data showed that the addition of ZA to macrophage-inducing culture conditions significantly inhibits the upregulation of F4/80, MHCII and CD11c. In addition, these data reveal that addition of tumour supernatant leads to polarisation of the macrophage phenotype towards M2, and that ZA can prevent this polarisation Next, we investigated whether treatment with ZA had an effect on survival. Mice were i.p. inoculated with AC29 tumour cells and treated daily with s.c. ZA injections or PBS as a control. The dosing schedule was chosen according to the literature, in which it was proven effective and non-toxic (P=0.2183)   . No signctively) . Long-tectively) .Taken together, although no significant differences on tumour progression and survival could be observed between untreated mice and mice treated with ZA, a reduction in the number of macrophages and an increase in the number of immature myeloid cells was detected.highGr-1high MDSC (PMN-MDSC), mononuclear CD11bhighGr-1int MDSC (MO-MDSC) and the CD11bhighGr-1low-MDSC  or PBS as a control. Mice were killed 25 days after tumour injection.As we observed a difference in the number of macrophages and immature myeloid cells during ZA treatment, we next wanted to know whether ZA treatment also affects macrophage phenotype +Gr-1+ cells was found in the spleen and effusion fluid of tumour-bearing mice of both groups. A significant increase was found in MO-MDSC both in the spleen and in the effusion fluid from the peritoneal cavity , whereas no significant differences were found in the proportion of PMN-MDSC and Gr-1low-MDSC . The proportion of macrophages in the spleen of ZA-treated mice was significantly lower compared with PBS-treated mice (P=0.0091). There was a trend towards a shift in macrophage phenotype from M2 to M1; however, the reduction in M2 macrophages was not significant (P=0.18). However, we found that the expression levels of CD206, expressed as the MFI, were significantly lower on M2 macrophages of ZA-treated mice compared with M2 macrophages in the spleen of PBS-treated mice (P=0.0095) (P=0.75). However, a significant increase in M1 macrophages was found, while M2 macrophages were reduced (P=0.035 and 0.33). A shift from M2 macrophage to M1 macrophage phenotype was found based on the MFI expression of CD206, confirming the findings in the spleen (P=0.14). As a result, a difference in ratio of M1:M2 was found (P=0.011) (An increase in CD11b=0.0095) . No signP=0.011) .β in the effusion fluid of ZA-treated mice . In addition, we observed a significant reduction in VEGF and CCL-2 (MCP-1) in the effusion fluid of ZA-treated animals (P=0.05 and 0.039). Levels of IL-10 and TNFα were not detectable in the effusion fluid.Enzyme-linked immunosorbent assay on the effusion fluid was performed to determine whether the reduction on CD206 expression on macrophages in ZA-treated mice was accompanied by changes in cytokine, chemokine and growth factor production. A significant increase was found in the levels of IL-6, IL-12 and IL-1β  levels and a significant increase in the levels of IL-6, IL-12 and IL-1β .Many effects of bisphosphonates have been reported in literature. Treatment with ZA prolongs the 1-year survival rate in breast cancer, prostate cancer and bladder cancer  was found on macrophages in tumour-bearing mice treated with ZA. Changes in M2-associated cytokines, chemokines and growth factors were observed in the effusion fluid after ZA treatment. We showed that ZA lowered the levels of VEGF in the effusion fluid of tumour-bearing mice. The reduction in VEGF and MMP-9 expressions by macrophages under the influence of ZA has been described to prevent tumour neovascularisation  due to treatment with ZA. Recently, studies have reported the role of MDSC in tumour progression. MDSC are most known for their immune suppressive function and induction of tumour-specific T-cell tolerance facilitating tumour immune escape . Therefoet al. reported similar observation. In their study, repolarisation of the macrophage phenotype was most promising during early tumour development, resulting in prolonged tumour-free survival and overall survival. In addition, they notify that shifting the phenotype in a host with a large tumour burden can be hazardous and no effects of ZA administration were observed when in situ carcinomas had progressed. From their data, it appears that tumour development is inhibited when the induction of M2 macrophages can be post-poned. We observed similar findings: shifting the phenotype from M2 to M1 at later stages of tumourigenesis seems less effective. Therefore, it can be hypothesised that preventing the formation of M2 macrophages is a critical key in preventing tumour outgrowth during early stages of disease development.A second reason as to why ZA has no effect on survival may be assigned to the shift in macrophage phenotype. As most studies showed that TAMs are polarised to an M2 phenotype that contribute to carcinogenesis, shifting the phenotype of macrophages to M1 seems promising. M1 macrophages produce less tumour growth-promoting soluble factors and can potentially kill tumour cells. However, the question rises whether shifting the phenotype in full-blown tumours is possible and whether repolarisation is as promising as believed previously . NegativA third explanation for the limited effect on survival could be that ZA has effects on processes during tumour development that do not have a dominant role in our tumour models. Most investigations are carried out in tumours with high metastasis rates in combination with other therapeutic approaches (like adjuvant endocrine therapy) . In thesN-telopeptide/creatinine ratio) and preliminary evidence of efficacy on skeletal-related events; however, no studies were performed comparing dose–effect relations on the tumour microenvironment. Although there are some concerns on side effects caused by ZA at higher levels (To study the effects of ZA on macrophages in the primary tumour, dose levels are higher than clinically advised. Doses selected for clinical trials were based on changes in bone resorption markers (including serum COOH-terminal telopeptide and urinary In conclusion, our data show that daily administration of ZA in tumour-bearing mice inhibits myeloid differentiation and shifts the balance from M2 macrophage phenotype to M1 macrophage phenotype. However, as a consequence the number of immature myeloid cells remains high in the spleen and in the effusion fluid, especially in the MO-MDSC population. This MO-MDSC population was found to have an immunosuppressive effect to which we ascribe the fact that survival was not improved with ZA in our study.


Sixty-three biopsies were from primary tumours, and eight were from recurrences. Overall plating efficiency ranged from 0.005 to 1.60% with a median of 0.052%. The majority of the colonies obtained from the biopsies were fibroblast marker-positive; the proportion of tumour marker-positive colonies ranged from 1 to 88% with a median of 15%. The median overall SF2 was 0.47 (range 0.24–0.96), the median tumour cell SF2 was 0.50 (range 0.11–1.0) and the median fibroblast SF2 was 0.49 (range 0.24–1.0). Comparing data from independent experiments, the overall SF2 was significantly correlated with the SF2 of fibroblasts (2P = 0.006) but not with the tumour cell SF2. The tumour cell and fibroblast radiosensitivities measured in the same individuals were not correlated . This finding seems to preclude a strong correlation between the radiosensitivity of tumour cells and fibroblasts. Concerning the clinical characteristics, neither of the measures of tumour radiosensitivity was correlated with T- and N-category, stage, tumour size, sex and age. However, the tumour cell radiosensitivity decreased with increasing grade of histopathological differentiation (2P = 0.012). The same tendency was found in two independent analyses of the same patient material. This correlation was not significant in case of the overall SF2 or the fibroblast SF2. © 1999 Cancer Research CampaignThe aim was to characterize the variation in the cellular in vitro radiosensitivities in squamous cell carcinomas of the head and neck, and to test for a possible correlation between different measures of radiosensitivity and the clinical and histopathological data. Cellular in vitro radiosensitivities were assessed in tumour biopsies from 71 patients using the modified Courtenay–Mills soft agar clonogenic assay combined with an immunocytochemical analysis. Radiosensitivity was quantified as the surviving fraction after a radiation dose of 2 Gy irrespective of cell type (overall SF


This paper is aimed at investigating how metastatic tumour growth influenced the haemostatic system of the host. Blood platelet count, blood fibrinogen level, the activated partial thromboplastin time (APTT) and the prothrombin time (PT) were determined at various intervals during growth and metastasis of a murine fibrosarcoma (mFS6) or one of its sublines with different metastatic capacity. Progressive thrombocytopenia and increase in fibrinogen level were observed during development of the tumour in all the animal groups studied, irrespective of the metastatic potential of the various sublines. No significant changes were observed in the PT or APTT values. These data support the concept that primary rather than metastatic growth influences the haemostatic system of tumour-bearing animals.


Nuclear magnetic resonance (NMR) proton imaging and measurements of the parameters T1 and T2, have been carried out in vivo on the murine mammary tumour T50/80. Tumours had been treated 24 h previously by photodynamic therapy . Proton images clearly demarcated a high signal-intensity region on the side of the tumour closest to the incident light beam, while the parts of the tumour more remote from the beam resembled the images from untreated controls. Both T1 and T2 values were raised in the high-intensity region. This high-intensity region was shown to correspond to PDT-induced histological necrosis, the low-intensity region to histologically intact tumour. Linear regression analysis of the relationship of depth of necrosis measured histologically and 'depth of necrosis' measured from the NMR images, yielded a slope of 0.93 (r2 = 0.95).


Tumour development is a multi-step process during which genetic and epigenetic events determine the transition from a normal to a malignant cellular state. In the past decade, extensive effort has been made not only to define the molecular mechanisms underlying progression to malignancy but also to predict the development of the disease and to identify possible molecular targets for therapy. Common to most tumours, several regulatory circuits are altered during multistage tumour progression, most importantly, the control of proliferation, the balance between cell survival and programmed cell death (apoptosis), the communication with neighbouring cells and the extracellular matrix, the induction of tumour neovascularization (angiogenesis) and, finally, tumour cell migration, invasion and metastatic dissemination. De-regulation of each of these processes represents a rate-limiting step for tumour development and, hence, has to be achieved by tumour cells in a highly selective manner during tumour progression.In this review we summarize recent advances in cancer research that have provided new insights in the molecular mechanisms underlying the transition between one tumour stage and the next and into their concerted action during tumour progression. Cultured human tumour cell lines as well as transgenic and knock-out mouse models of tumorigenesis have been instrumental in these experimental approaches. © 2000 Cancer Research Campaign


Four novel zinc (II)-substituted phthalocyanines, varying in charge and hydrophobicity, were evaluated in vivo as new photosensitizers for photodynamic therapy. Two rat tumours with differing vascularity were used: a mammary carcinoma (LMC1) and a fibrosarcoma (LSBD1), with vascular components six times higher in the latter (10.8%+/-1.5) than in the former (1.8%+/-1.4). Each sensitizer was assessed for tumour response relative to normal tissue damage, and optimum doses were selected for further study, ranging from 0.5 to 20 mg kg(-1). Interstitial illumination of the tumours was carried out using a 200-microm-core optical fibre with a 0.5 cm length of diffusing tip, at either 680 or 692 nm, depending on the sensitizer. Light doses of between 200 and 600 J were delivered at a rate of 100 mW from the 0.5-cm diffusing section of the fibre. Maximum mean growth delays ranged from 9 to 13.5 days depending on sensitizer and type of tumour, with the most potent photosensitizer appearing to be the cationic compound. Histopathological changes were investigated after treatment to determine the mechanism by which tumour necrosis was effected. The tumours had the appearance of an infarct and, under the conditions used, the observed damage was shown to be mainly due to ischaemic processes, although some direct tumour cell damage could not be ruled out.


The derivation of the median volume (MV) and the geometric standard deviation (SDg) for a suspension of tumour cells quantifies the size and distribution of tumour cell aggregates in the suspension. Data collected in a group of 14 experiments shwoed a significant correlation of 0.80 (P less than 0.001) between the number of lung tumours formed by a suspension of B16 melanoma cells injected i.v. into C57BL/6J mice and the product of the MV and SDg of each cell suspension. These data define a size parameter of tumour cell suspensions that correlates with the intravascular survival properties of tumour cells.


Intralesional γ-inulin (0.1 mg mouse−1) delivered immediately after PDT rivaled zymosan  in delaying the recurrence of B16BL6 melanomas. This effect of γ-inulin was further enhanced by IFN-γ pretreatment. Tumour C3 protein levels, already elevated after individual PDT or γ-inulin treatments, increased much higher after their combination. With fibrosarcomas MCA205 and FsaR, adjuvant γ-inulin proved highly effective in reducing recurrence rates following PDT using four different photosensitisers . At 3 days after PDT plus γ-inulin treatment, over 50% of cells found at the tumour site were CTLs engaged in killing specific targets via perforin–granzyme pathway. This study demonstrates that γ-inulin is highly effective PDT adjuvant and suggests that by amplifying the activation of complement system, this agent potentiates the development of CTL-mediated immunity against PDT-treated tumours.Host response elicited by photodynamic therapy (PDT) of cancerous lesions is a critical contributor to the clinical outcome, and complement system has emerged as its important element. Amplification of complement action was shown to improve tumour PDT response. In search of a clinically relevant complement activator for use as a PDT adjuvant, this study focused on  Photodynamic therapy (PDT), in which the destruction of cancerous lesions is achieved by the targeted site-localised generation of reactive oxygen species formed by the transfer to molecular oxygen of energy captured by the light excitation of photosensitising drugs , has notSaccharomyces cerevisiae) is a standard and potent complement activator but it is not safe for clinical use . The present study reports that γ-inulin treatment is highly effective in elevating PDT-mediated cures of mouse tumours and reveals that its action amplifies CTL activity against PDT-treated tumours.Supportive evidence of the importance of the contribution of the activated complement system includes the decrease in cure rates of PDT-treated tumours following blockage of complement activation by the inhibitor FUT-175 , or spec6 cells mouse−1 subcutaneously at the sacral region on the dorsal site. The tumours were treated when their largest diameter reached 6–8 mm (around 8 days post implant).Melanoma B16BL6  polyfructofuranosyl α-D-glucose, in which an unbranched chain of up to 100 fructose moieties is linked to a single terminal glucose (μl) of γ-inulin suspension immediately after PDT light exposure. Controls involving the intratumoural injection of the same volume of saline verified the absence of injection volume-related unspecific effects.The  glucose . The for glucose . The treThe photosensitisers used in this study were benzoporphyrin derivative monoacid ring A , Photofrin , m-tetrahydroxyphenylchlorin , and chlorin e6 . They were injected intravenously at 24 h , 3 h (BPD), or 2 h (ce6) before photodynamic light treatment.−2 (power output measured by a light power metre divided by the surface of the illuminated area encompassing the treated tumour). During light treatment, mice were restrained unanaesthetised in special holders designed to expose the sacral region of their backs.The procedure and equipment used for photodynamic light treatment was described in detail elsewhere  were suspended in PBS and injected intratumourally  immediately after PDT light treatment. Mouse IFN-γ  was administered peritumourally (3 × 104 U mouse−1) at 24 h before PDT light treatment.For zymosan treatment, particles derived from the cell wall of After therapy, mice were monitored for tumour growth and no sign of palpable tumour at 90 days post treatment qualified as a cure. The effect of adjuvants in the absence of PDT was tested by following tumour growth rates determined by caliper measurement of the lesion's three orthogonal diameters. Saline only controls were tested for all of the used agents and they verified the absence of unspecific effects related to injections.γ-inulin treatment. The content of complement C3 protein in tumour tissue homogenates was determined using enzyme-linked immunosorbent assay (ELISA) assay as previously described in full detail  were subjected to standard enzymatic disaggregation procedure  was used as isotype control for CD107a. These samples were then analysed by three-colour flow cytometry using the Coulter Epics Elite ESP , with 20 000 cells included for each test. The percentage of FITC-positive cells was determined in populations gated as CD45+CD8+.Tumours excised at 3 days after rocedure . Single-P<0.05.Tumour response/cure results were statistically analysed using Log-rank test. The differences between means of the data from other experiments were statistically evaluated based on Mann–Whitney test. Statistical differences were considered significant when −1) given immediately after PDT light treatment doubled the time for tumour recurrence, while over three-fold increase in the recurrence time was observed with γ-inulin (0.1 mg mouse−1) applied in the same fashion (γ-inulin treatment without PDT showed no detectable impact on tumour growth (not shown). No benefit was found with increasing the γ-inulin dose in conjunction with PDT treatment to 0.2 mg per mouse  to the treatment protocol. This IFN-γ treatment, which by itself was not effective in boosting PDT response, produced in the combination with γ-inulin on average over a four-fold prolongation in the post PDT recurrence time of B16BL6 tumours . The results show that γ-inulin treatment alone produced a rise in tumour C3 levels at 2 h after injection with a similar level found 4 h later  at 6 h post therapy.To verify the impact of  h later . A compaγ-inulin as adjuvant to PDT was further examined with different tumour models and photosensitisers. The treatment of MCA205 fibrosarcomas with BPD-based PDT initially ablated all the lesions, but this was in most cases followed by the recurrence within the next 2 weeks and only 25% of mice remained tumour-free at 90 days post therapy (γ-inulin (0.1 mg mouse−1 i.t.) immediately after photodynamic light treatment showed a dramatic improvement evident as a decreased tumour recurrence compared to the PDT only group and high levels of cures. Similar results with the same tumour model were obtained with two different photosensitisers, Photofrin and ce6; in both cases, the chosen PDT only dose was in the moderate/halfway cure range and the adjuvant γ-inulin treatment increased the level of tumour cures to fully or highly curative range by reducing the recurrence rate .The efficacy of  therapy . The gronce rate . This efve range . Similarγ-inulin is revealed by the flow cytometry-based detection at the treated tumour site of cytotoxic T cells that have lysosomal-associated membrane protein-1  detectable on their surface (CD8+CD107a+), which occurs when these cells undergo degranulation (releasing granzyme B and perforin) upon their contact with specific target cells  giving on average over 50% degranulating cytotoxic T cells per total collected cell population  to greatly increase their efficacy in enhancing the proliferation of antigen-specific T cells  while this increase with zymosan was only two-fold  is well recognised for its capacity to stimulate phagocytic activity of macrophages and dendritic cells, and for augmenting the ability of these cells for antigen processing and presentation by increased quantity and diversity of peptides presented on their surface in the context of MHC  is a highly efficient adjuvant to tumour PDT with mouse tumour models. As its adjuvant efficacy was documented with different tumour types, including poorly immunogenic melanoma model, it can be expected that γ-inulin will work as adjuvant to PDT of human cancers. The results suggest that by boosting the activation of complement system, this agent potentiates the development of cytotoxic T-cell-mediated immunity against the PDT-treated tumour, which results in the abolishment or reduction in tumour recurrence rates.In summary, the present study demonstrates that the specific potent complement activator


Two groups of rats without tumour were treated with resection of a normal liver lobe or ILT of normal liver. A challenging tumour was implanted into the liver of each rat 2, 5 or 10 weeks after primary treatment. Rats were killed 6, 12 and 48 days (or earlier due to their condition) after challenge . Immunohistochemical techniques were used to determine lymphocytes  and macrophages  in rats having had treatment of a primary tumour. Interstitial laser thermotherapy of the first tumour was followed by eradication of challenging tumour and absence of tumour spread. This contrasted with rapid growth and spread of challenging tumour in the other groups. In the challenging vital tumour tissue and in the interface between the tumour and surroundings, the number of ED1 macrophages and CD8 lymphocytes was higher in rats having been treated with the ILT of tumour than in those having undergone resection of the tumour-bearing lobe. The number of ED2 macrophages and CD4 lymphocytes was low and did not vary between these two groups. Interstitial laser thermotherapy elicited an immune response that eradicated a challenging tumour and was associated with increased numbers of tumour-infiltrating macrophages and CD8 lymphocytes.Previous studies in our laboratory have shown that interstitial laser thermotherapy (ILT) of an experimental liver tumour is superior to surgical excision, at least partly due to a laser-induced immunological effect. The aim of the present study was to investigate the time–response relationship of the ILT-induced immunisation and the cellular response of macrophages and lymphocytes. A dimethylhydrazine-induced adenocarcinoma was transplanted into the liver of syngeneic rats. Rats with tumour were treated 6–8 days later (tumour size 0.25–0.40 cm In the future, curative treatment of solid tumours is likely to consist of local treatment  coupled with different types of adjuvant therapy. The reason why local excision alone may not be sufficient for cure is the risk for occult disseminated disease and, also, the risk for local recurrence. Surgery is known to induce immunosuppression and the release of growth factors that may stimulate growth of residual tumour , but complementary studies were performed with Wistar FU from our own breeding. Rats weighed 224–292 g at the time of tumour implantation. They were housed individually and had free access to standard food pellets  and tap water ry, Lund . The tumTumour implantation was performed with vital and macroscopically homogenous tumour. At midline laparatomy and using an operating microscope , a 3-mm-long and 2-mm-deep incision was made on the anterior surface of the liver. A piece of Spongostan  was applied to the incision area for 5 min to control bleeding. A piece of the tumour (1.0 mm in diameter) was then implanted into the parenchyma, followed by application of Spongostan for another 2 min. The first tumour (the tumour to be treated) was implanted into the left lateral lobe of the liver, whereas the second  tumour was implanted into the median lobe of the liver. Tumour preparations were made immediately before transplantation and were kept on ice cubes until implanted.Rats were anaesthetised with an intraperitoneal injection of 5% chloral hydrate (0.5 mg per 100 g body weight) for tumour implantation. Ether was used for all other procedures that required anaesthesia, including treatments, challenges and euthanasia. All treatments were performed using a midline abdominal incision. Treatment procedures were followed by intramuscular injection of buprenorphine , 0.002 mg per 100 g body weight, for analgesia.Animal procedures were performed according to the recommendations of the Swedish Board of Animal Research and were approved by the Committee of Animal Ethics at Lund University.3 and did not vary between the treatment groups (P>0.05). A challenging tumour was implanted into the liver of each rat 2, 5 or 10 weeks after primary treatment (groups I–IV). Rats were planned to be killed 6, 12 and 48 days after challenge . However, rats were euthanised as soon as they showed signs of inactivity or distress. At that time, there was usually palpable intraabdominal tumours.We used 288 rats which were randomly allocated to four groups. Two groups of rats with tumour (I–II) were treated with ILT of tumour (I) or resection of the tumour-bearing lobe (II). Two groups of rats without tumour (III–IV) were treated with resection of a normal liver lobe (III) or ILT of normal liver (IV). Treatment of rats with tumour (I–II) was performed 6–8 days after implantation, at which time tumour had a size of 140±25 (mean±s.e.m.) mmAfter killing, the liver, lungs, peritoneum and all tissues containing suspect tumour growth were harvested for microscopic examination. Immunohistochemical examinations were carried out after challenge in rats having had treatment of a primary tumour (groups I–II).μm flexible bare fibre at a laser output power of 2 W. The bare fibre was placed at right angles into the centre of the tumour, at a depth measuring one-third of the thickness of the liver, including the tumour. A feedback thermistor probe  was placed perpendicularly into the liver parenchyma at a distance of 3 mm from the tumour margin and at a depth of 2 mm from the liver surface. The distance from the tip of the bare fibre to the thermistor probe averaged 6.4±0.1 (mean±s.e.m.) mm.Thermotherapy was carried out with a system consisting of an Nd:YAG laser and a temperature feedback control unit interfaced with the laser, as described in detail elsewhere , 1998 F. The lasInterstitial laser thermotherapy was performed at a steady-state target temperature of 46°C for 30 min at the thermistor probe, that is, 3 mm from the tumour margin. The treatment characteristics were based on previous studies defining the temperature and duration of treatment needed for complete tumour necrosis, as judged 6 days after treatment , 1998. IResection of the tumour-bearing, or normal, left lateral lobe was performed after division of the falciform ligament and the triangular plicae on the left side. A ligature of 4-0 Suturamid  was tied around the most central part of the venous and arterial pedicle to the left lateral liver lobe. The liver was transected with a pair of scissors 1–2 mm distal to the ligature and 10.3±0.4  mm proximal to the tumour, as measured macroscopically. A soft gauze was carefully placed under the left lateral liver lobe in order to collect possible bleeding from the resection line.M sodium phosphate buffer (PB) as a rule for 24 h. The livers with tumours were delivered uncut to the pathologist, who examined the specimens while unaware of the treatment. A section was cut through what was considered to be the largest diameter of the lesion(s), sometimes supplemented with one or two additional sections. The specimens were then rinsed, dehydrated and embedded in paraffin. After sectioning, the slides were processed for immunohistochemistry or stained with haematoxylin/eosin.Liver tissues were fixed in 4% paraformaldehyde in 0.1 The sizes of total tumour and necrotic tumour were measured under the microscope with a micrometer, because it was impossible to distinguish necrosis, inflammatory changes and tumour with the naked eye, especially in rats that had been treated with laser thermotherapy.M sodium phosphate-buffered saline (PBS) with 0.25% Triton X-100, pH 7.2. Immunohistochemical reaction for tissue macrophages was performed with ED1 and ED2 mouse anti-rat antibodies (diluted 1 : 200) , using a secondary rabbit anti-mouse antibody (ad modum Mayer. Negative controls (buffer instead of primary antibody) were performed to each reaction.The sections were deparaffinised with xylene and brought to 96% alcohol and washed in 0.1 Labelled cells were counted per visual field with an × 40 objective and an × 10 ocular. As a rule, only whole fields were counted on peripheral vital tumour and granulation tissue. It was tried to count six fields for each type of tissue, but that was not always possible. For each rat, ED1, ED2, CD4 and CD8 slides were counted one after the other to ensure that the same areas were counted with all antibodies. The order of examination of slides varied. Necroses and large vessels were avoided. The mean per visual field was used in the statistical analysis.V) was estimated according to the formula V=a × b2/2, where a is the largest width and b is the maximum diameter perpendicular to the width of the tumour . The difference between the ILT and resection groups was more obvious for CD8 lymphocytes: numbers were larger in the ILT group both at 6 and 12 days after challenge, and both in the tumour capsule and in viable tumour . ED2 and CD4 cells were always few, and there were no differences between the ILT and resection groups (P>0.05) (The number of ED1 macrophages in tumour capsule and viable tumour was significantly larger in the ILT (group I) than in the resection groups (II) at 12 days after challenge ((P>0.05) . There wAs a rule, ED1- and ED2-positive cells had the appearance of macrophages in vital tumour . In the Local tumour therapy with hyperthermia focuses on two different aspects: (a) atraumatic reduction or ablation of tumour . This stγ or TNF-α helps to generate cytolytic effector cells.CD8 lymphocytes and ED1 macrophages invaded the challenging tumour. This agrees with findings in studies in which antitumour immunity has been induced and where these two cell types have been found to be of importance . AntitumThough we and the cited authors have used a syngeneic tumour model, it can be argued that the tumour was raised a long time ago, in our system 30 years ago. A genetic drift may have occurred, in the tumour and/or the rats, and transplantation antigens may have been responsible, at least in part, for the outcome. However, in our previous twin tumour study, the same effect was obtained in rats that were inoculated with a pure tumour cell suspension at two different sites in the liver  are involved in mediating the immunological response against tumours. Heat shock protein–tumour peptide complexes are taken up by antigen-processing cells like macrophages/dendritic cells, where the tumour peptides are processed and presented to T cells, eliciting an immune response . In our The reason why we use the low temperature of 46°C 3 mm from the tumour margin (during 30 min) is that we want to obtain cell death that develops into necrosis within a time range of hours to a few days (In conclusion, laser thermotherapy appears to be a promising method for curative treatment of solid tumours. Hyperthermia may play an important role in immunotherapeutic approaches in the future.


In mice with a transplantable mammary carcinoma, treatment with the prostaglandin-synthesis inhibitors flurbiprofen or indomethacin produced various beneficial effects. Survival time after excision of the transplanted tumour was increase, particularly when the drugs were given with the chemotherapeutic agents methrotrexate and melphalan, and there were more disease-free survivors. The combined treatment with flurbiprofen also gave less tumour recurrence at the excision site. Flurbiprofen did not seem to alter the bioavailability of the chemotherapeutic agents.


WT1 located on chromosome 11p13, FWT1 on 17q12-q21, and FWT2 on 19q13. Two out of 19 Wilms tumour families evaluated showed strong evidence against linkage at all three loci. Both of these families contained at least three cases of Wilms tumour indicating that they were highly likely to be due to genetic susceptibility and therefore that one or more additional familial Wilms tumour susceptibility genes remain to be found. © 2000 Cancer Research CampaignThree loci have been implicated in familial Wilms tumour:


The presence of tumour cells in the circulation may predict disease recurrence and metastasis. To improve on existing methods of cytological or immunocytological detection, we have developed a sensitive and quantitative technique for the detection of carcinoma cells in blood, using the reverse transcriptase polymerase chain reaction (RT-PCR) identifying transcripts of the pancarcinoma-associated tumour marker EGP-2 (KSA or 17-1A antigen). The amount of EGP2 mRNA was quantified using an internal recombinant competitor RNA standard with known concentration and which is both reversely transcribed and co-amplified in the same reaction, allowing for a reliable assessment of the initial amount of EGP2 mRNA in the sample. Calibration studies, seeding blood with MCF-7 breast carcinoma cells, showed that the assay can detect ten tumour cells among 1.0 x 10(6) leucocytes. The PCR assay revealed that normal bone marrow expresses low levels of EGP2 mRNA, although immunocytochemistry with the anti-EGP2 MAb MOC31 could not identify any positively stained cell. Analyses using this RT-PCR assay may prove to have applications to the assessment of circulating tumour cells in clinical samples.


In the Funding section a digit was missing from one of the grant numbers. NIH grant U54-CA11296 should read U54-CA112967.


Isolated perfusion of the extremities with high-dose tumour necrosis factor α (TNF-α) plus melphalan leads to dramatic tumour response in patients with irresectable soft tissue sarcoma or multiple melanoma in transit metastases. We developed in vivo isolated organ perfusion models to determine whether similar tumour responses in solid organ tumours can be obtained with this regimen. Here, we describe the technique of isolated kidney perfusion. We studied the feasibility of a perfusion with TNF-α and assessed its anti-tumour effects in tumour models differing in tumour vasculature. The maximal tolerated dose (MTD) proved to be only 1 μg TNF-α. Higher doses appeared to induce renal failure and a secondary cytokine release with fatal respiratory and septic shock-like symptoms. In vitro, the combination of TNF-α and melphalan did not result in a synergistic growth-inhibiting effect on CC 531 colon adenocarcinoma cells, whereas an additive effect was observed on osteosarcoma ROS-1 cells. In vivo isolated kidney perfusion, with TNF-α alone or in combination with melphalan, did not result in a significant anti-tumour response in either tumour model in a subrenal capsule assay. We conclude that, because of the susceptibility of the kidney to perfusion with TNF-α, the minimal threshold concentration of TNF-α to exert its anti-tumour effects was not reached. The applicability of TNF-α in isolated kidney perfusion for human tumours seems, therefore, questionable. © 1999 Cancer Research Campaign


Cells from two adenocarcinomas, an adenoma and a metastatic node were isolated in soft agar. Expression of antigens, CEA, Y haptenic blood group and 791T-p72, defined by a range of candidate antibodies for tumour targeting was assessed. All of the cells expressed low levels of CEA but high levels of the Y haptenic blood group antigen although there was enormous inter and intraclonal variation. Of particular interest was the membrane expression of 791T-p72 antigen on all of the dividing tumour cells as previous studies had shown that 791T/36 antibody reacted with tumour stromal elements rather than malignant cell surfaces. The DNA content was abnormal in all of the cells whether they were derived from diploid or aneuploid primary tumours. They all grew readily in athymic mice and at least one monoclonal antibody, 791T/36, localised efficiently within these xenografts. Clonogenic cells therefore expressed the three tumour-associated antigens, several at higher levels than observed in the primary tumour. Monoclonal antibody 'cocktails' should therefore allow antibody mediated drug cytotoxicity to be effective at eradicating rapidly dividing tumour cells.


Phorbol-12-myristate-13-acetate (PMA), calcium ionophore A23187 and platelet activating factor (PAF) stimulated the generation of oxygen free radicals (nitro-blue tetrazolium reduction) in Ehrlich ascites tumour (EAT) cells. PAF was effective at an optimal concentration of 4 μM, but was inhibited by BN 52021, a specific PAF antagonist. Lyso-PAF was ineffective. Inclusion of different lipids during incubation prior to the addition of PAF, resulted in the activation/inhibition of free radical generation. Among the phospholipids at a concentration of 50 μg/ml, the order of activation was phosphatidylserine > phosphatidylglycerol > phosphoinositides > phosphatidylinositol > phosphatidylethanolamine. Phosphatidylcholine was not effective, while sphingolipids were inhibitory. In addition, Ehrlich ascites tumour cells grown in mice under marginal vitamin A deficiency, showed an augmented production of free radicals compared to control cells. This was suppressed by exogenous addition of vitamin A or superoxide dismutase. These results suggest that membrane lipids and dietary factors like vitamin A probably function as physiological modulators in regulating the free radical generation.


However, repeat dearterializations for 60 min in portal deviatedrats induced irreversible liver damage and all rats died in a few days. Repeated dearterializationsfor 60 minutes alone retarded the tumour growth as efficiently (p = 0.007). Simultaneousocclusion of the hepatic artery and the portal vein for 30 minutes with a side-side porto-cavalshunted  did not affect tumour growth (p = 0.154). Liver aminotransferases(ASAT and ALAT) were substantially increased following dearterialization for 30 min inrats with either an end-side or a side-side porto-caval shunt. Dearterialization for 60 min in ratswith end-side porto-caval shunts gave a further release of ASAT and ALAT.As liver tumours receive some of their blood supply from the portal vein, we wanted to illustratethe influence of portal blood flow in combination with dearterialization in the treatment of livertumours. Forty male, inbred Wistar/Furth rats with an adenocarcinoma transplanted to the liverwere treated with various inflow occlusions repeated daily for 5 days. Deviation of the portalblood flow alone with an end-side porto-caval shunt did not alter the tumour growth (		In conclusion, portal deviation did not augment the therapeutic benefit of repeat dearterializationsfor the treatment of this experimental liver tumour. Repeat dearterializations aloneseemed to be a feasible and efficient therapy for liver tumours.


One hundred aspirations using a fine needle have been performed on 94 patients with a suspected diagnosis of malignant tumour, 31 of which were in patients with recurrent tumour. In 90 aspirates where histology was also available there was agreement between histological and cytological diagnosis in 81 (90%). This percentage was identical when only previously undiagnosed tumours were considered (60). In 4 aspirates no cells were obtained from tumours in which a diagnosis was made histologically and in 5 there was disagreement with histology, either regarding the presence of malignancy, or tumour type. The technique of fine needle aspiration is simple, rapid, safe and reliable. It is particularly valuable when emergency treatment is required, necessitating a very rapid diagnosis, or when the tumour is entirely intra-abdominal and the patient is unfit for laparotomy. Repeat aspirates may be performed to assess progress following treatment, or multiple suspected tumour sites may be aspirated to assist staging. The technique may be used to confirm the presence of relapsing tumour. Aspiration cytology may prove valuable as a further dimension in the interpretation of histological sections in a variety of childhood tumours, and in some circumstances may be sufficient in itself to establish a diagnosis.


Corresponding dose-effect curves obtained for growth of the primary tumour (Pr) and its metastases in unirradiated tissues showed that local irradiation of Pr caused an exponential decrease in growth of metastases due to any dissemination occurring after irradiation, but was also responsible for stimulating growth of metastases already established before treatment in lymph nodes and in lungs. This stimulating effect was most marked when Pr was larger at the time of treatment and when high doses were given to eradicate Pr. This effect is attributed to the liberation of growth stimulating substances (GSS) from a pool of GSS produced in the irradiated Pr by sterilized, but metabolically active and growing tumour cells (HR cells). This effect of HR cells on tumour growth and metastases was also demonstrated when rats were inoculated with viable tumour cells and subsequently treated by injecting large doses of HR cells prepared The systemic effects of GSS on metastases were most clearly seen after immunosuppression of recipient hosts by sublethal whole body irradiation, since immunosurveillance in unirradiated rats resulting from a rapidly developing allogenic tumour-host incompatibility caused marked reductions in clonogenicity of the tumour which tended to overshadow the GSS effect. The latter was also masked in immunosuppressed hosts when excessively high rates of dissemination were due to growth of large Pr inocula for sufficiently long to “saturate” the capacity for growth of metastatic tumour in lymph nodes and lungs.The relevance of these findings to clinical radiotherapy is discussed.


The use of photodynamic therapy (PDT) as an adjunct to curative tumour resection was investigated in a tumour recurrence model, using rat mammary adenocarcinoma BN472. Tumours were inoculated subcutaneously in 60 animals and resected after 21 days of growth. Immediately after removal, the operation site was exposed to 320-450 nm light of 0.1 W cm-2 and 60 J cm-2 after photosensitisation with either Photofrin (5 mg kg-1 i.v. 48 h before illumination) or 5-aminolaevulinic acid (ALA) (2 mg ml-1 in drinking water for 9 days). Porphyrin concentrations were measured in tissue samples. After 28 days, animals treated with adjunctive PDT had a significantly longer tumour-free interval than controls (P < 0.01); median 25 days (Photofrin), 18 days (ALA), 14 days (controls). Moreover, in the PDT groups significantly fewer rats had lymph node metastasis. A prophyrin concentration ratio between tumour and mammary tissue of 2:1 was found after Photofrin and 4:1 after ALA. The results indicate that adjuvant intraoperative PDT may be a safe and effective method of destroying residual tumour, thereby preventing locoregional tumour recurrence.


Postoperative deaths and neurological injury have resulted from hyponatraemia associated with the use of hypotonic saline solutions following surgery. We aimed to determine the rates and types of intravenous fluids being prescribed postoperatively for children in the UK.A questionnaire was sent to members of the British Association of Paediatric Surgeons (BAPS) and Association of Paediatric Anaesthetists of Great Britain and Ireland (APAGBI) based at UK paediatric centres. Respondents were asked to prescribe postoperative fluids for scenarios involving children of different ages. The study period was between May 2006 and November 2006.The most frequently used solution was sodium chloride 0.45% with glucose 5% although one quarter of respondents still used sodium chloride 0.18% with glucose 4%. Isotonic fluids were used by 41% of anaesthetists and 9.8% of surgeons for the older child, but fewer for infants. Standard maintenance rates or greater were prescribed by over 80% of respondents.Most doctors said they would prescribe hypotonic fluids at volumes equal to or greater than traditional maintenance rates at the time of the survey. A survey to describe practice since publication of National Patient Safety Agency (NPSA) recommendations is required. Several publications in the last decade describe the syndrome of hospital acquired hyponatraemia in children -3. The uThe danger of hyponatraemia is cerebral oedema resulting in seizures, neurological deterioration and sometimes in tentorial herniation and death. This can cause permanent neurological injury and death . HyponatFollowing a hyponatraemic death associated with the use of sodium chloride 0.18% with glucose 4% in a child with gastroenteritis, the Royal College of Anaesthetists issued a bulletin following a request from Royal College of Paediatrics and Child Heath, advising caution with its use . As the A postal questionnaire was sent to members of the BAPS and APAGBI working at tertiary referral centres in the UK. The study period was between May and November 2006. The questionnaire asked the respondent to document the type and volume of postoperative fluids prescribed in the following clinical scenarios.1. An elective excision of an abdominal duplication cyst in a 6 kg, six-month-old baby.2. A 28 kg, nine-year-old child following an uncomplicated open appendicectomy.3. A 9 kg, nine month old infant following an urgent laparotomy for an intussusception.The questionnaire stated that all three children were well perfused and haemodynamically stable at the end of surgery. For each case the participant was asked what type of fluid they would prescribe for the first 24 hours postoperatively, and at what infusion rate.Standard maintenance rates were defined as 100 mls per kilogram for the first 10 kilograms of body weight, 50 mls per kg for the next 10 kg and 25 mls per kg thereafter per 24 hours  correspoA total of 364 questionnaires were sent. We had a 51% response rate from 120 forms sent to Paediatric surgeons and a 47% response rate from 244 forms sent to anaesthetists. We excluded 15 questionnaires from anaesthetists which had not been completed – the respondents stating that in their institution surgeons wrote or altered all postoperative fluid orders. Prescription orders varied widely. Most respondents used commercially available solutions although 3% made up their own fluid by adding dextrose to standard pre-prepared solutions.The type of fluid prescribed is shown in table Overall, standard maintenance rates were prescribed by 63% of anaesthetists and surgeons as shown in table Despite widespread publicised concerns, over 20% of anaesthetists and 38% of surgeons still prescribe the very hypotonic sodium chloride 0.18% with glucose 4%. This is putting large numbers of children at risk of hyponatraemia and its devastating consequences. However with recent NPSA guidelines and trust actions of withdrawal of all sodium chloride 0.18% solution, this should be avoided.Most prescriptions were for standard maintenance rates based on the original work of Holliday and Segar almost 50 years ago . Their ctypically prescribed and at what rate.The scenarios chosen were typical for a tertiary care paediatric surgical department and were uncomplicated to avoid complex answers. Abdominal cases were chosen as other types of paediatric surgery do not necessarily require postoperative fluids. Postoperative fluid therapy prescriptions are often written according to a departmental policy. We therefore only asked what fluids are More difficult scenarios may have prompted more complex and individually tailored prescriptions that would have obscured our objectives. Minimal blood loss would be anticipated for all cases, but the urgency of surgery and potential for on-going third space and gastrointestinal losses varied. This was to determine if anticipated losses prompted a different fluid prescription both in tonicity and quantity.The simplified nature of the questionnaire did not allow the patient to be regularly assessed and treatment tailored accordingly. This would have reflected actual practices more accurately. However, with this in mind space was allocated on the questionnaire for further comments. Most respondents who provided additional information detailed the importance of regular review, monitoring of haemodynamic status and blood glucose. Only four mentioned sodium specifically. Reassuringly all fluid boluses prescribed to replace nasogastric losses or to improve perfusion were with isotonic solutions.Worryingly between 8 and 38.8% of respondents gave maintenance fluid at volumes far greater than standard rates, presumably to compensate for blood loss, third space or gastrointestinal losses. As these losses are isotonic and the maintenance fluid was usually hypotonic, this practice is particularly dangerous.Also alarming is the apparent inability of some respondents to recall the maintenance formula correctly. 5% of anaesthetists and 18% of surgeons multiplied the entire weight by 100 mls/kg/day. This was of no consequence for the younger children who were less than 10 kg, but grossly overestimated the volume of fluid required for the 28 kg child. Such errors put children further at risk of hyponatraemia. The heavier the child, the more significant this error becomes.Given the warning from two Royal Colleges and widespread published concerns from a number of authors calling for the use of sodium chloride 0.18% with glucose 4% to be revised ,13-17, wThose concerned argue that hypotonic fluids are appropriate, but at lower rates of infusion ,11,12. WBased on current evidence and NPSA guidelines sodium chloride 0.18% solutions should be abandoned as a maintenance fluid for children in the post operative period.Children with anticipated additional fluid losses should have them replaced with isotonic solutions only (sodium chloride 0.9% or Hartmans). Those at particular risk of hypoglycaemia  should receive glucose containing solutions. All fluid boluses should be 20 ml/kg of isotonic fluid or 2 aliquots of 10 ml/kg over a short time period. As there is no 'one size fits all' prescription, the importance of regular review with electrolyte monitoring cannot be overestimated.The authors declare that they have no competing interests.PD participated in the data analysis and drafted the manuscript. TH carried out the postal survey, participated in data analysis and helped to draft the manuscript. TA conceived of the study, participated in its design, statistical analysis and coordination. KL conceived of the study, participated in its design and was the main coordinator. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


It has been demonstrated that neutrophils are indispensable for its anti-tumour effectiveness. We decided to evaluate the extent of the anti-tumour effectiveness of PDT combined with administration of granulocyte colony-stimulating factor (G-CSF) as well as the influence of Photofrin®and G-CSF on the myelopoiesis and functional activity of neutrophils in mice. An intensive treatment with G-CSF significantly potentiated anti-tumour effectiveness of Photofrin®-based PDT resulting in a reduction of tumour growth and prolongation of the survival time of mice bearing two different tumours: colon-26 and Lewis lung carcinoma. Moreover, 33% of C-26-bearing mice were completely cured of their tumours after combined therapy and developed a specific and long-lasting immunity. The tumours treated with both agents contained more infiltrating neutrophils and apoptotic cells then tumours treated with either G-CSF or PDT only. Importantly, simultaneous administration of Photofrin®and G-CSF stimulated bone marrow and spleen myelopoiesis that resulted in an increased number of neutrophils demonstrating functional characteristics of activation. Potentiated anti-tumour effects of Photofrin®-based PDT combined with G-CSF observed in two murine tumour models suggest that clinical trials using this tumour therapy protocol would be worth pursuing. © 2000 Cancer Research CampaignPhotofrin


Isolated disseminated tumour cells (DTC) are regarded as surrogate markers for minimal residual disease in breast cancer. Characterisation of these cells could help understand the known limitations of adjuvant therapy. Of particular interest is their oestrogen-receptor (ER) status because endocrine adjuvant therapy remains a cornerstone of breast cancer treatment.Bone marrow (BM) aspirates from 254 patients with primary breast cancer were included in this study. A double immunofluorescence staining procedure was established for the identification of cytokeratin (CK) positive/Erα-positive cells. ERα status of the primary tumour was assessed immunohistochemically using the same antibody against ERα.6 mononuclear cells. DTCs demonstrated ERα positivity in 12% of the patients. The ERα expression was heterogeneous in 10 of the 38 (26%) patients with more than one DTC. The concordance rate of ERα status between primary tumour and DTC was 28%. Only 12 of 88 patients with ERα-positive tumours also had ERα-positive DTCs.In 107 of 254 (42%) breast cancer patients, CK-positive cells could be detected in the BM. More than one DTC in the BM was observed in 38 of the 107 patients. The number of detected cells ranged between 1 and 55 cells per 2 × 10Primary tumours and DTCs displayed a concordant ERα status in only 28% of cases. Most of the DTCs were ERα negative despite the presence of an ERα-positive primary tumour. These findings further underline the distinct nature of DTCs and may explain the failure rates seen in conventional endocrine adjuvant therapy. Tumour cell dissemination is a common phenomenon in breast cancer where isolated disseminated cells can be detected in up to 40% of patients at the time of primary diagnosis -3. BasedMore detailed knowledge about their cellular and molecular characteristics could help define a targeted secondary adjuvant therapy in patients with primary breast cancer who have undergone conventional adjuvant therapy. It has already been shown that about 40% of DTCs express human epidermal growth factor receptor 2 (HER2) and that in some patients with recurrent breast cancer their HER2 status may differ from that of the primary tumour . Since tPrior to any therapy, between 10 and 20 ml of bone marrow were aspirated from the anterior iliac crest of 254 primary breast cancer patients undergoing surgical treatment from 2005 to 2007 at the Department of Gynecology and Obstetrics, University Hospital Tuebingen, Germany.4Cl, 10 mM KHC03, 0.1 mM EDTA pH 7.2). Using a cytocentrifuge , 106 mononuclear cells were spun onto a glass slide. The slides were air-dried overnight at room temperature. For detection and characterisation of DTCs, slides were fixed in a 0.5% neutral buffered formalin solution for 10 minutes. Control cytospins with ERα-positive MCF-7 cells were prepared, stored and fixed in the same way to ensure that ERα negativity of a patient's sample was not due to a handling error. Two slides per patient was analysed for the presence of DTCs (2 × 106 cells per patient).The characteristics of the patients are shown in Table For establishing the ERα staining procedure, preparations of breast cancer cell lines MCF-7 and SKBR3 mixed with either BM or peripheral blood mononuclear cells (PBMCs) from a healthy volunteer were used Figure . To optiAfter an initial washing step with PBS , cells were blocked for 30 minutes with normal goat serum  at a 1:10 dilution. The automated double immunofluorescence staining procedure was performed on the DAKO Autostainer using the monoclonal rabbit ERα-antibody SP1  for 60 minutes and secondary detection with a goat anti-rabbit antibody, labelled with Alexa Fluor 594  for 30 minutes. Cytospins were then incubated with a pan-cytokeratin (CK) antibody (C11) directly conjugated to fluorescein isothiocyanate (FITC)  for 30 minutes. This monoclonal antibody recognises human CKs 4, 5, 6, 8, 10, 13 and 18. Counterstaining was performed with 4'6-diamidino-2-phenylindole (DAPI) in mounting media . Preparations of the breast cancer cell line MCF-7 mixed with PBMCs from a healthy volunteer served as a positive control for CK and ERα staining. ERα negative control slides of SKBR-3/PBMC mixtures were also included with each batch of samples. Cytospins of PBMCs with no added tumour cells served as a negative control for both.Slides were manually analysed for the presence of tumour cells using a computerised fluorescence microscope Axiophot . To screen for ERα-positive tumour cells, a single-pass filter for individual fluorochromes, FITC, Texas Red or DAPI, and a dual-pass filter for FITC/Texas Red were used. Criteria for evaluation of immunostained cells were based on the criteria of the International Society of Hematotherapy and Graft Engineering Working group for standardisation of tumour cell detection and the consensus statements ,11. CritImmunohistochemical analysis was performed either on core biopsies or surgical resection specimens. The tissue was fixed in 4.5% buffered formalin (pH 7.0) and embedded in paraffin. Immunohistochemical staining was performed on 3 to 5 μm thick sections using a commercially available ABC kit . The ERα antibody (clone SP1) was diluted 1:200 in Tris-HCl (pH 7.5) and applied according to the manufacturer's instruction . 3,3'diaminobenzidine (DAB) was used as a chromogen. Finally, the slides were counterstained with haematoxylin and mounted for examination. For assessment of the ERα status, the percentage of cells with nuclear reactivity  and the intensity of ER staining  was determined. ERα expression was scored semi-quantitatively using the Remmele-score (score nuclear staining × score intensity of ER staining). Tumours with a score of 2 or more were considered ERα positive.A chi-squared test or Fisher's exact test was used to evaluate the relation between ERα-positive DTCs and clinicopathological factors. Statistical analysis was performed by SPSS, version 11.5 . p < 0.05 was considered statistically significant.6 mononuclear cells). In 38 of the 107 (35%) BM-positive patients, more than one DTC could be detected. No correlation was observed between positive BM status and any of the established prognostic markers including the ERα status of the primary tumour  had ERα-negative tumour cells in BM Table . ERα-posOf the 107 patients, 38 had more than one DTC in the BM. Of these 38 patients, 28 had only ERα-negative tumour cells Table .The ERα status of the primary tumour could be determined in all 107 patients with detectable DTCs in the BM. ERα positivity of the primary tumour was demonstrated in 88 82%) of these patients. The concordance rate between ERα status of DTCs and primary tumour was 28%. Only 12 of the 88 (14%) patients with ERα-positive primary tumour had ERα-positive DTCs in the BM. In contrast, 18 of 19 (95%) patients with ERα-negative primary tumours also had ERα-negative DTCs  could increase the number of patients eligible for endocrine therapy and on the other hand  could explain why endocrine therapy fails in a subset of hormone receptor-positive patients.et al. found that only two of 11 patients with ERα-positive primary tumours (18%) had ERα-positive DTCs [et al. investigated the ERα status of circulating tumour cells in metastatic breast cancer patients and their corresponding primary tumours: fourteen of 16 patients (88%) had ERα-positive primary tumours, but only three patients had ERα-positive circulating tumour cells [Looking at a large patient group, our data confirms findings of previous, smaller studies, indicating that the ERα status of the primary tumour does not necessarily reflect the ERα status of minimal residual disease Table . In an oive DTCs . Reuben ur cells . Our reset al. compared the HER2 status of DTC and primary tumour in 137 cases [et al. reported HER2-positive circulating tumour cells in nine of 24 (38%) patients with recurrent breast cancer who had HER2-negative tumours [As mentioned above, these discrepancies between DTCs and the primary tumour are not confined to ERα-expression: Solomayer 37 cases  and foun tumours . It has  tumours ,19.Different hypotheses need to be discussed with regard to our findings. One possible explanation is the clonal heterogeneity of the primary tumour: ERα-negative cells could be more likely to disseminate, corresponding to the worse prognosis of predominantly ERα negative tumours and – inversely – to the demonstrated decreased invasiveness and metastatic potential of ERα-expressing breast cancer cells ,21. MCF-One interesting hypothesis currently under discussion is the theory that some or all DTCs, the presumed precursor cells of systemic metastatic disease, are in fact cancer stem cells. As recently published, this theory states that tumour growth and formation of secondary tumours can be traced to a small subpopulation of tumour cells, so called cancer stem cells ,30. FirsThe phenotypic discrepancies between DTCs and their corresponding primary tumours have the potential to increase our understanding of why treatments are successful in some, but not in other patients, paving the way towards more individualised forms of treatment. The target of adjuvant therapy is the eradication of minimal residual disease. In order to optimise treatment strategies, the phenotypic properties of DTCs – the surrogate marker of minimal residual disease – should be taken into account in addition to characterisation of the primary tumour. Already, the available studies looking at phenotypic properties of DTCs have often found them to be non-proliferative, ERα negative and HER2 positive ,16,28. FBM: bone marrow; BSA: bovine serum albumin; CK: cytokeratin; DAB: 3,3' diaminobenzidine; DAPI: 4'6-diamidino-2-phenylindole; DTC: disseminated tumour cells; ER: oestrogen receptor; FITC: fluorescein isothiocyanate; HER2: human epidermal growth factor receptor 2; MHC: major histocompatibility complex; PBMC: peripheral blood mononuclear cellsThe authors declare that they have no competing interests.TF, GPB, SD, NK, AS and ES made substantial contributions to the conception and design of the study, acquisition of data, and analysis and interpretation of data. TF, SB, HS and HN were involved in drafting the manuscript or revising it. All authors read and approved the final manuscript.


A study of the healing of peritoneal defects in the rat in the presence of the Walker 256 tumour has been made. The healing process was investigated histologically, by autoradiography, and by hydroxyproline estimation of the healing peritoneal wound. There was no difference in the rates or quality of the healing process in the control or tumour bearing animals.


The anti-inflammatory agent sodium aurothiomalate appears to act upon mononuclear phagocytes, inhibiting their lysosomal enzyme activity. Evidence is presented that gold salts can increase the number of lung tumour nodules that develop following intravenous injection of tumour cells and pretreatment can enhance the take of a subcutaneous tumour inoculum. In contrast, they do not affect the later growth of tumour. Gold salts can also suppress the action of systemically administered C. parvum in inhibiting the growth of subcutaneous tumours. These results are taken as supporting the evidence in favour of a fast acting nonspecific anti-tumour mechanism, probably macrophage mediated, that can be inhibited by gold salts and enhanced by C. parvum. The effect of gold salts upon other biological changes induced by C. parvum is examined, including its adjuvant action, and the results are discussed in the context of the mechanisms underlying the immunotherapeutic action of this organism.


Extensive experience with isotransplants of 27 different tumours , all of strictly spontaneous origin in laboratory bred mice of low cancer strains CBA/Ht and WHT/Ht, has revealed no evidence of tumour immunogenicity. Of approximately 20,000 maintenance transplants, none failed and none regressed; of almost 10,000 carefully observed tumours arising from small or minimal inocula of tumour cells, none spontaneously regressed. The number of injected viable tumour cells required to give a 50% probability of successful transplantation (the TD50) ranged from approximately 1 cell to greater than 10,000 cells among the 27 tumours; high TD50 values, which were dramatically reduced by various procedures having no immunological significance, did not signify active "resistance" of the hosts. In the case of all of 7 randomly selected tumours, prior "immunization" of recipients with homologous lethally irradiated cells increased their tumour receptivity. Several experiments using various tumours failed to give evidence that immunity could be non-specifically induced or that a massive preponderance of lymphocytes from specifically sensitized mice could inhibit tumour transplantation or growth in vivo; no trace of "resistance" to tumour was adopted by isogeneic recipients of lymphocytes from regional nodes of tumour bearers. A limited review of the recent literature on tumour immunity shows that practically all the animal data presented in support of a general theory of tumour immunogenicity or to provide a basis for active clinical immunotherapy have been obtained from transplanted tumour systems which entail artefactual immunity associated with viral or chemical induction of the tumours or their allogeneic transplantation. It is suggested that isotransplants of spontaneously arising tumours are the only appropriate models of human cancer and that any genuine rapport between the animal laboratory and the clinic requires their exclusive use.


Phyllodes tumours and angiosarcoma are both rare mesenchymal tumours. There are no reports of their coexistence in theliterature except in families with germline p53 mutations. Here we report a case of an elderly woman who developed anextensive angiosarcoma of the scalp nearly 4 years after surgical removal of a borderline malignant phyllodes tumour of thebreast. The scalp lesion was initially thought more likely to be a metastasis of her first rare tumour than a second equally rareprimary tumour, but histologically this was not the case. The case and the literature are discussed.


Tumour-specific antiserum was raised in sheep against idiotypic determinants on the surface immunoglobulin of neoplastic lymphocytes from a patient with chronic lymphocytic leukaemia (prolymphocytic variant). The complement-activating IgG1 subclass of the anti-idiotype was prepared from the serum in monodisperse form for infusion. Two treatments of 480 and 1200 mg caused the white-cell count to fall by one-third and one-half respectively. However, there was a rapid resurgence, so that by 8 days after each treatment the counts were restored to approximately 85% of their former levels. No change was noted in the size of spleen or lymph nodes. Each treatment probably destroyed 4-8 X 10(11) cells, some 10% of the total tumour load. The antibody was rapidly consumed, and there was evidence of heavy utilization of complement.


Electron spin resonance (ESR) spectroscopy was used to examine changes in the concentration of paramagnetic metal ions in Yoshida tumours carried by female Wistar rats. Blood, spleen and lymph nodes from these animals were also examined by ESR. A decrease in the concentration of a paramagnetic species associated with mitochondrial activity, and marked increases in those thought to be associated with inflammatory or immune reactions and cell lysis, were observed in the tumours within one day of implantation. During development of the tumour, and during its regression after treatment with methylene dimethane sulphonate (MDMS), further changes were observed in the concentration of the species. These were dependent on the region of the tumour examined. In blood, development of the tumour produced an increase in ceruloplasmin and a decrease in iron-transferrin. An increase in spleen weight, as the tumour developed, was accompanied by a small decrease in the concentration of species with g-values of 6-0 and 4-3, which was reversed on regression of the treated tumour. The presence of metastases in the regional lymph nodes produced distinguishable changes in the ESR spectra.


Detection of loss of heterozygosity (LOH) is usually performed on homogenised tumour specimens. In this type of analysis samples with a low percentage of tumour cells have to be excluded and possible intra-tumour heterogeneity is obscured. In this study we report the application of polymerase chain reaction (PCR)-driven LOH detection with in total 22 microsatellite markers for chromosome 1q, 3p, 3q, 4p, 6p, 6q, 11p, 11q, 17p, 17q, 18p, 18q, Xp and Xq on flow-sorted cells from fresh and paraffin-embedded ovarian tumour tissue. Titration experiments showed that LOH can be detected with as few as 100 cell equivalents of DNA. Clear examples of LOH could be detected in the sorted aneuploid fractions from one unilateral and two bilateral ovarian tumours from three patients. In two samples the sorted fraction was less than 10% of the total sample. The bilateral tumours from the same patient showed loss of identical alleles for one marker (case OV64) and two markers (case OV69), indicative of their monoclonal origin. Multiparameter flow cytometry using two different ovarian tumour markers (MOv18 and BMA180), an anti-cytokeratin monoclonal antibody (MAb) (M9), an anti-vimentin MAb (V9) and a MAb against the panepithelial antigen 17-1A on the fresh ascites cells of the fourth ovarian cancer patient was used to investigate possible intra-tumour heterogeneity. We showed the presence of at least three phenotypically different populations, of which the diploid, keratin-positive, vimentin-negative population showed a similar LOH pattern as the aneuploid population (DNA index = 1.7), indicative of its neoplastic origin. The same LOH pattern was shown in an omentum metastasis from this patient also having the same aneuploid DNA index of 1.7. The sharing of the same LOH pattern by the diploid and aneuploid tumour cell populations suggests that the observed allele loss events occurred before the development of aneuploidy. PCR on flow-sorted cells is thus an important tool to study clonal diversity in tumours.


Fas-Lribozyme-carrying cells exhibited slightly enhanced growth rate and less degree of spontaneous apoptosis in vitro as compared with vector controls. In nude mice, Fas-Lribozyme-carrying cells grew faster with lesser apoptosis, formed bigger tumour with significantly fewer infiltrating cells in the tumour area, and triggered relatively milder tumour-associated liver damage than vector controls did. Thus, down-regulation of Fas-L not only improved viability of glioma cells but also reduces local immune responses that may consequently affect tumour formation. Taken together, our findings imply that endogenous expression of Fas-L in malignant cells is not always growth promoting. © 2001 Cancer Research Campaign  http://www.bjcancer.comFas-L  expresses in a variety of tumours and has been proposed to play a role in tumour formation and metastasis. The contribution of Fas-L to tumour growth, however, is not conclusive especially in systems using cells with over-expressed Fas-L. In this study we down-regulated the expression o Fas-L in human glioma cells by a hammerhead ribozyme (Fas-L


Blood vessel staining (CD31) and quantification of the vessels as well as TAMs and tumour-associated dendritic cells (TADCs) in the A673 model showed reduction rates of 85 to >94%, even 9 days after the end of therapy. In addition, CD11c+ TADCs, which have been shown to potentially differentiate into endothelial-like cells upon stimulation by tumour released growth and differentiation factors, were similarly reduced by clodrolip or antibody treatment. These results validate clodrolip therapy in combination with angiogenesis inhibitors as a promising novel strategy for an indirect cancer therapy aimed at the haematopoietic precursor cells that stimulate tumour growth and dissemination and as a tool to study the role of macrophages and dendritic cells in tumorigenesis.Tumour-associated macrophages, TAMs, play a pivotal role in tumour growth and metastasis by promoting tumour angiogenesis. Treatment with clodronate encapsulated in liposomes (clodrolip) efficiently depleted these phagocytic cells in the murine F9 teratocarcinoma and human A673 rhabdomyosarcoma mouse tumour models resulting in significant inhibition of tumour growth ranging from 75 to >92%, depending on therapy and schedule. Tumour inhibition was accompanied by a drastic reduction in blood vessel density in the tumour tissue. Vascular endothelial growth factor (VEGF) is one of the major inducers of tumour angiogenesis and is also required for macrophage recruitment. The strongest effects were observed with the combination therapy of clodrolip and a VEGF-neutralising antibody, whereas free clodronate was not significantly active. Immunohistologic evaluation of the tumours showed significant depletion of F4/80 Solid tumours are not only composed of malignant cells, but are complex organ-like structures comprising many cell types, including a wide variety of migratory haematopoietic and resident stromal cells . The rolβ), the angiopoietins (Ang1 and Ang2), interleukins such as IL-1 and IL-8, tumour necrosis factor-α (TNF-α), thymidine phosphorylase (TP), the matrix metalloproteinases MMP-9 and MMP-2, nitric oxide (NO) and chemokines  produce a vast number of factors that promote tumorigenesis. The most prominent include basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), transforming growth factor beta , human A673 rhabdomyosarcoma (CRL-1598) were from American Type Culture Collection (ATCC) and HUVE cells from PromoCell . The cell culture media DMEM and HUVEC growth medium were from Gibco, Paisley, UK. Peritoneal macrophages were freshly isolated from Sv129 mice. Female Sv129 mice and CD1 nude mice were from Charles River Wiga  and kept in standard housing and normal diet at the animal facility of the Paul Scherrer Institute. Animal studies were approved by the Veterinary Department of the Canton Aargau, Switzerland and performed under the licenses (No. 75533 and 75534) issued to RA Schwendener. The ethical guidelines that were followed meet the standards required by the UKCCCR guidelines  corresponding to 1 : 0.3 : 0.01 mol parts were prepared by freeze–thawing and filter extrusion. The dry lipid mixture was solubilised in a physiologic phosphate buffer  supplemented with mannitol (230 mM) and 2.64 g clodronate . The resulting multilamellar vesicles were freeze–thawed in three cycles of liquid nitrogen and water at 40°C, followed by repetitive (5–10 ×) filter extrusion through 400 nm membranes  using a Lipex™ extruder . Nonencapsulated clodronate was removed by dialysis . Liposome size and homogeneity were routinely measured with a Nicomp laser light scattering particle sizer . Routinely prepared small unilamellar clodrolip liposomes contain approximately 20 mg clodronate ml−1 and have a mean diameter of 135 ± 55 nm.Clodronate liposomes, termed clodrolip, were essentially prepared as described before (in vitro cytotoxicity of clodronate was assessed as described before (−1) and cell viability was determined by addition of WST-1 reagent  according to the manufacturer's recommendations.The 6 50 μl−1) or A673 rhabdomyosarcoma cells  were injected subcoutanously (s.c.) on the flanks of mice. Treatment was started 6 h after inoculation of F9 cells  and 24 h after inoculation of A673 cells , respectively. The mice (6–8/group) received clodronate dissolved in phosphate buffer  or clodrolip by intraperitoneal (i.p.) injection as initial dose of 2 mg 20 g−1 mouse body weight, followed by 1 mg for the subsequent doses. The Abs were given at 0.5 mg 20 g−1 mouse body weight in 100 μl PB by intravenous (i.v.) injection into the tail vein. Tumour growth was measured in a blinded fashion with a caliper and volumes were calculated using the equation: V=πab2/6 . Relative percentual tumour growth was normalised to day one. Mice were killed 8–22 days after onset of treatment and tumours and spleens removed for histology.Exponentially growing F9 teratocarcinoma  assays were carried out as described previously (164 (Canis familiaris) and VEGF-E (originating from the orf-family of pox viruses) were produced in Pichia pastoris and purified by affinity chromatography as described previously . Recombinant mVEGF164 (purity >95%) was from ReliaTech. The synthetic ETH-2 human scFv antibody phage library was from D Neri   were coated with 25 μg ml−1 mVEGF164 in PB (first and second panning) followed by hVEGF165 (third panning) for 12 h at room temperature. Selected scFv antibodies were screened by ELISA for binding to recombinant hVEGF165 and mVEGF164 using the M2 anti-FLAG-antibody  for detection. The formation of (scFv′)2 Ab dimers was accomplished by introduction of C-terminal cysteines as described  (164 and cfVEGF164 of SZH9 were determined with a competitive ELISA assay (125I) using the Iodogen method. Female CD1 nude mice bearing subcutaneously implanted A673 cells (6–8 × 106) at each flank were injected with 3 μg (1.85 × 105 MBq) scFv Abs in 0.1 ml phosphate buffer . Mice (three/group) were killed at 0.17, 0.5, 1, 2, 4, 6 and 24 h after injection. Antibody concentrations in blood, liver, kidneys and tumours were determined and expressed as percent of injected dose per gram tissue (%ID g−1). Pharmacokinetic parameters were calculated with GraphPad Prism 4 software.Recombinant cfVEGFt-test. All P-values were calculated vs PB controls unless indicated otherwise. P-values of <0.05 were considered as statistically significant. The quantification of the IHC data was performed with the image analysis software analySIS .All measurements are shown as average±s.e.m. Statistical analysis of data from growth inhibition experiments was performed using a two-tailed, unpaired in vitro. Treatment with clodrolip killed peritoneal macrophages in a concentration-dependent manner resulting in an IC50 of 2.8 mM or 1 mg ml−1 , murine F9 teratocarcinoma and human A637 rhabdomyosarcoma cells  mg ml−1 . HUVEC, ml−1  (P=0.0185) was obtained with an early treatment on days 0, 4, 8 and 12. Clodronate (5 mg) given on days 0, 4, 8, 12 had an insignificant inhibitory effect of 45% (P=0.21), comparable to the days 4, 8 and 12 delayed clodrolip schedule . The delayed onset of therapy at time points where tumours were already established and well vascularised (day 4 or 8) resulted in less pronounced growth inhibition, suggesting that macrophage depletion in large tumours is not sufficient to efficiently inhibit tumour growth.We next studied the effects of clodrolip on tumour progression and angiogenesis in the highly vascularised and fast-growing syngeneic F9 teratocarcinoma mouse tumour model. Mice were treated i.p. with PB, empty liposomes, clodronate or clodrolip. The initial clodronate dose was 2 mg 20 g and 12) . The mosP=0.028) was obtained with HD clodrolip application, whereas clodronate HD therapy was less active . Interestingly, clodrolip plus clodronate combination therapy (LD+LD) was equally active  as clodrolip LD alone , suggesting that clodronate LD was ineffective. Quantification of macrophage depletion and blood vessel density by immunohistochemistry in A673 tumours revealed a superior, but dose-dependent depletion by clodrolip compared with clodronate therapy. To assess the depletion effects, four to five tumour sections from individual mice per treatment group were analysed by determining positively stained areas (pixel counts transformed to percent) in three defined regions of interest of identical size. As shown on the bar graph in + macrophages are more susceptible to clodrolip than the quantitatively larger population of the F4/80+ TAMs. Clodrolip was also more efficient in reducing CD31+ EC counts than clodronate . Clodrolip treatment did not cause overt toxic side effects in the F9 and the A673 mouse tumour model and we assume that the spleen is repopulated with macrophages after the end of therapy within 1–3 weeks. Likewise, the tumour stroma may be repopulated from the periphery within the same time frame. Compared to PB-treated control mice, treatment with a total dose of 5 mg of either clodronate or clodrolip did not significantly change body weight (<±5%) during the monitored time periods  and low dose . As shown in iods see . This suP=0.003). Blocking VEGF with V65 antibody reduced tumour volumes by 65% (P=0.047). The combination protocol resulted in a significantly higher reduction of 92% (P=0.001), corresponding to 10 and 28% higher values compared to the single treatments, respectively.Recognising that clodrolip-mediated depletion of TAMs might not be effective enough for complete suppression of tumour growth, we combined clodrolip with an antiangiogenic therapy by systemic application of anti-VEGF antibodies. In the F9 model, mice were either treated with PB, an unspecific control antibody A1, with the anti-mVEGF Ab V65 . ClodrolEncouraged by the results obtained with V65, we reasoned that a VEGF-blocking antibody that neutralises both tumour cell-secreted human VEGF and mouse VEGF expressed by stromal host cells, such as macrophages, ECs and fibroblasts, would be advantageous to prevent the growth of xenograft tumours. Thus, we generated a mouse–human crossreactive anti-VEGF scFv recombinant antibody SZH9 and functionalised it at its carboxy terminus to allow further derivatisation and dimerisation   and treatment with SZH9 by 59% (P=0.04). The combination therapy was again clearly better than the single treatments resulting in growth inhibition by 74% , tumour blood vessels were virtually undetectable and significant reduction in vessel density was observed even 9 days later  and of MOMA1+ macrophages by 90% (P=0.0001) in clodrolip-treated animals. Interestingly, even single treatment with SZH9 resulted in depletion of F4/80+ by 52% (P=0.18) and of MOMA1+ macrophages by 30%  after SZH9, by 59% (P=0.033) after clodrolip and by 74% (P=0.029) after clodrolip plus SZH9 treatment, respectively (+ TADCs by 96% (P=0.043) after SZH9, by 99% (P=0.0034) after clodrolip and by 99% (P=0.0006) after clodrolip plus SZH9 treatment, respectively, was observed , clodrolip alone by 89% (P<0.0001), whereas the combination therapy gave an 85% (P<0.0001) reduction which was statistically not significantly different from clodrolip monotherapy (+ and MOMA1+ macrophage density vs microvessel counts (CD31+ cells) showed a clear separation of tumours treated with clodrolip or clodrolip plus SZH9 compared to tumours treated with SZH9 alone or with A1 or PB. (+ and CD11c+ cell depletion with vessel density (CD31+ cells) confirms these results  induced by IL-4, IL-13 (M2a) or IL-10 (M2c) and glucocorticoid hormones. Differential cytokine and chemokine production and coordinated temporal and spatial activities of these cells in the tumour stroma are key features of polarised macrophages and promote tumour angiogenesis and growth . However+ TADC subsets apparently did not exhaust all stromal sources of proangiogenic and tumourigenic mediators, such as for example mast cells, neutrophils, fibroblasts and other DC subsets, and possibly also pericytes. Thus, TAM depletion, as most antiangiogenic therapies, ought to be regarded as an adjuvant treatment in combination with conventional chemo- or radiotherapy. Low-dose clodrolip therapy applied at higher frequency, also termed ‘metronomic’ therapy  either suppress regulatory T cells (Treg) or lead to T-cell unresponsiveness (+ TAMs. These TAMs are able to inhibit a T-cell-mediated immune response controlled by STAT1 (in vivo ablation of CD11c+ dendritic cells in diphtheria-toxin transgenic mice abrogates priming of cytotoxic T-lymphocyte precursors in immune responses to cell-associated antigens, a phenomenon called cross-priming (+ DCs, termed vascular leucocytes. These DCs simultaneously express both endothelial and dendritic cell markers, and, depending on the microenvironment, assemble into functional blood vessels or act as antigen-presenting cells. Dendritic cell precursor-mediated vasculogenesis has also been found to be regulated through the cooperation of β-defensins and VEGF-A, where VEGF-A primarily induces their endothelial-like specialisation and migration to blood vessels (The important role of VEGF was further supported by the distinct antiangiogenic effect obtained with the anti-VEGF antibody monotherapy, which resulted in a reduction of F4/80siveness . Immaturby STAT1 . Moreove vessels . Colony  vessels  and blocRecently, it was shown by Finally, VEGF-C production by TAMs was proposed to play a role in lymphangiogenesis and lymphatic metastasis in several human cancers (Taken together, our findings provide solid evidence for the importance of TAMs, and possibly also of TADCs, in the establishment of a microenvironment favouring tumour growth and dissemination. Clodronate- or other bisphosphonate liposome-mediated macrophage depletion regimens open new possibilities to study the role of tumour infiltrating cells, for example by gene expression profiling of TAM-depleted tumours. In addition, TAM depletion combined with new antiangiogenic or cytotoxic therapies is a promising new approach with high clinical potential.


The technique of labelled mitoses was used to examine cell proliferation within grafts of human colonic and rectal tumours in immune deprived mice. Most of the data were obtained on the first passage but in some cases up to the third passage was used. It was found to be difficult to obtain precise kinetic data on this type of tumour material, but the results did allow some estimates to be made, particularly of the duration of the G2 and S phases of the mitotic cycle. The average G2 duration was 6 h and the average S phase was 14 h. It is concluded that whilst xenografts may differ in a number of respects from the tumour in the patient, they nevertheless constitute a type of experimental tumour that is worthy of further study.


The use of 5-aminolaevulinic acid (ALA) is gaining increasing attention for photosensitization in photodynamic therapy of superficially localized tumours. The aim of this work was to determine the kinetics of porphyrin generation in tissues after topical application of ALA delivered in different vehicles on the skin overlying the tumour and normal skin of mice. Maximal accumulation was found in tumour 3 h after ALA application in both cream and lotion preparations. Normal and overlying tumour skin tissues showed different kinetic patterns, reflecting histological changes when the latter is invaded by tumour cells. Liver, kidney, spleen and blood porphyrins also raised from basal levels, showing that ALA and/or ALA-induced porphyrins reach all tissues after topical application. During the first 24 h of ALA topical application, precursors and porphyrins are excreted by both urine and faeces. ALA lotion applied on the skin overlying the tumour induced higher accumulation of tumoural porphyrins than cream, and lotion applied on normal skin appeared to be the most efficient upon inducing total body porphyrins. This work has demonstrated the great influence of the formulation of ALA vehicle on penetration through the skin. Knowledge of the kinetics of porphyrin generation after different conditions of ALA application is needed for the optimization of diagnosis and phototherapy in human tumours. © 1999 Cancer Research Campaign


The effects were determined of an initial i.v. injection of 0.2 ml suspensions of 125IUdR-labelled lymphosarcoma cells on the early arrest patterns of a second injection of cancer cells into tumour-bearing mice. The results indicate that interactions between the first injection and the host markedly affected the arrest pattern of the second dose in the lungs, but not the livers, of tumour-bearing animals. These observations are explained on the basis of the injected fluid volumes, which are considerable in mice, in relation to their total blood volumes of approximately 2 ml.