IGR-NB8 is an immature NB xenograft with MYCN amplification and 1p deletion, which is sensitive to CPT-11. Athymic mice bearing advanced-stage subcutaneous tumours were treated with CPT-11 (27 mg kg−1 day−1 × 5) every 21 days (1 cycle) for a maximum of four cycles. After tumour regrowth, a new in vivo passage was performed and the CPT-11 treatment was repeated. After the third passage, a resistant xenograft was obtained (IGRNB8-R). The tumour growth delay (TGD) was reduced from 115 at passage 1 to 40 at passage 4 and no complete or partial regression was observed. After further exposure to the drug, up to 28 passages, the resistant xenograft was definitively established with a TGD from 17 at passage 28. Resistant tumours reverted to sensitive tumours after 15 passages without treatment. IGR-NB8-R remained sensitive to cyclophosphamide and cisplatin and cross-resistance was observed with the topoisomerase I inhibitor topotecan. No quantitative or qualitative topoisomerase I modifications were observed. The level of expression of multidrug resistance 1 (MDR1), MDR-associated protein 1 (MRP1) and, breast cancer resistance protein, three members of the ATP-binding cassette transporter family was not modified over passages. Our results suggest a novel resistance mechanism, probably not involving the mechanisms usually observed in vitro.CPT-11 (irinotecan) is a DNA-topoisomerase I inhibitor with preclinical activity against neuroblastoma (NB) xenografts. The aim was to establish  Irinotecan (CPT-11), a semisynthetic water-soluble analogue of camptothecin, belongs to a new family of anticancer drugs, the DNA-topoisomerase I inhibitors. Topoisomerase I is an essential nuclear enzyme that relaxes DNA torsional tension during fundamental processes such as replication, transcription, recombination and repair, and represent a target for many anticancer drugs. Irinotecan settles on the DNA-topoisomerase I complex (cleavable complex), stabilises it and inhibits the religation of DNA. Cytotoxicity arises during the replication process when the advancing replication fork and cleavable complex collide, leading to irreversible DNA damage and to the initiation of a series of events that result in cell death .MYCN gene amplification (MDR1 gene overexpression (Neuroblastoma (NB) is one of the most common solid tumours in young children and is responsible for approximately 7.5% of all cancer among children younger than 15 years of age . Cyclophosphamide was purchased from Asta-Medica , cisplatin from Bellon and etoposide from Novartis . Drugs were dissolved in a 0.9% sodium chloride solution immediately before injection on each day of treatment.Female specific pathogen-free Swiss athymic mice (6–8 weeks old) were bred in the Animal Experimentation Unit of the Institut Gustave-Roussy. Animals were housed in sterile isolators and fed with irradiated nutriments  and filtered water. Experiments were carried out under the conditions established by the European Community directive no. 86/609/.CEE and in accordance with the UKCCCR guidelines   .3 tumour fragments were xenotransplanted subcutaneously  into 50 athymic mice. On day 0 of the treatment, mice bearing a 100–300 mm3 subcutaneous tumour were randomly assigned to one treated and one control group of five to 10 mice each. Tumour perpendicular diameters were measured twice a week with a caliper, and tumour volume calculated according to the following equation: V (mm3)=(d2 (mm2) × D (mm))/2, where d and D are the smallest and largest perpendicular tumour diameters, respectively. Each group of mice was treated according to the average weight of the group. Animal body weights were recorded twice weekly and mortality was checked daily. The experiments lasted until tumour volumes reached 1500–2000 mm3.For each experiment, 15–30 mm−1 day−1 for 5 consecutive days. This dose was previously shown to induce 100% complete regressions (CR) and to be well tolerated (no treatment-related death and no body weight loss)  and PR as a tumour regression exceeding 50% of the initial tumour volume. At least two consecutive tumour measurements had to be observed in order to retain CR and PR. TGD was defined as the difference between the treated group and the control group, within the median time to reach a tumour volume that was five-fold the initial tumour volume (i.e. time to 5). For each tumour of the treated groups, the individual TGD was defined as the difference between the individual time to 5 and the median time to 5 of the control group. Since the duration of treatment was different from one passage to another, we also considered the TGD, corrected for the duration of treatment (TGDc), which was defined as the difference between TGD and the duration of treatment. TFS were defined as animals that were free of palpable tumour at the end of the experiment (at least 120 days).The activity of each drug was evaluated according to three criteria: (1) the number of complete (CR) and partial (PR) tumour regressions; (2) the TGD; (3) the number of tumour-free survivors (TFS) . CR was Xenograft tissue specimens were fixed in acetic acid–formalin–ethanol  and embedded in paraffin. The paraffin-embedded sections were stained with haematoxylin–eosin–saffranin for morphology.MYCN copy number was measured using the TaqMan 5′ nuclease fluorigenic real-time quantitative PCR assay, as reported previously . Hybridisation of differentially labelled tumour and normal DNA to normal metaphase chromosomes was performed using previously published methods  and homogenised with a potter teflon-glass homogeniser. NaCl (0.55 M) was added and incubated for 1 h on ice for total cell extraction. After centrifugation at 12 000 r.p.m. for 30 min, the supernatant was assayed for topoisomerase I activity. Protein concentration was determined by the BCA method (Pierce). Proteins (50 μg) were separated electrophoretically in 7.5% SDS–polyacrylamide gels and then transferred to a nitrocellulose membrane . Blots were incubated with human polyclonal topoisomerase I antibody (Topogen INC) diluted at 1 : 8300 followed by the anti-protein A horseradish-peroxidase-conjugated antibody (Amersham Pharmacia Biotech). Detection was performed using a chemiluminescence (ECL) enzyme immunoassay (Amersham Pharmacia Biotech).Crude extracts were prepared from NB xenografts. Frozen tissues (50 mg) were grossly minced, suspended in lysis buffer  was incubated with each diluted extract at 37°C for 30 min in 10 × Topoisomerase I assay buffer (Topogen Inc). DNA topoisomers were separated by gel electrophoresis in 1.25% agarose and stained with ethidium bromide. One arbitrary unit of topoisomerase I activity was defined as the amount of topoisomerase I showing relaxation of 0.25 μg DNA under the above-described conditions. Topoisomerase I activity was expressed in arbitrary units (a.u.) per mg of protein.Topoisomerase I catalytic activity of crude extracts was examined by a DNA relaxation assay using supercoiled pHOT1 plasmid DNA as the substrate . For each sample, 10 extracts were serially diluted in buffer containing 10 mMDR1 and MRP1 mRNA expression was analysed by RT–PCR as reported previously (μm thick sections and rehydrated. Sections were prepared with the Histo-mouse kit (Zymed) according to the manufacturer's instructions, and incubated for 1 h at room temperature with mouse anti-BCRP BXP-21 monoclonal antibody  diluted at 1 : 20. Detection was performed using a rabbit anti-mouse IgG horseradish peroxidase conjugate. A further analysis of BCRP expression was performed by Northern blotting. Total RNA was prepared using Trizol reagent, according to the manufacturer's instructions. In all, 20 μg of total RNA were fractionated on a 1% agarose–formaldehyde gel and subsequently transferred to a nitrocellulose membrane filter . Blots were prehybridised for 1 h at 42°C in 5 × SSC , 5 × Denhardt's solution, 0.2% SDS, 100 μg ml−1 salmon sperm DNA and 50% deionised formamide. The blots were then probed using 25 ng of the 32P-labelled BCRP/MXR/ABCP probe at 42°C overnight. After washing in 1 × SSC/0.1% SDS for 20 min at room temperature and three 10-min washes with 0.2 × SSC/0.1% SDS at 65°C, blots were analysed using a phosphor imaging system (Fujix Bas 2000).−1) was administered over the same period of time (68 days) and tumour growth displayed the same pattern. Complete regression and PR were observed after the first cycle of treatment. Tumour volumes remained stable during the following three cycles. When treatment was discontinued, tumours remained stable for about 1 month and then started to grow again. Over the first three passages, IGR-NB8 tumour response to CPT-11 was significantly reduced with a TGD from 115 to 69, that is, a TGD corrected for the duration of treatment from 47 days at the first passage to 1 day at the third passage .The influence of CPT-11 treatment on the growth of the IGR-NB8 xenografted tumour over 28 passages is shown in Figure 1−1 × 5) of treatment. As shown in Figure 2in vivo during the first eight passages was completely reverted after 15 growth passages of IGR-NB8-R without any further exposure to CPT-11. Thus, in vivo acquired resistance to CPT-11 was revertible.In order to evaluate the stability of the resistance acquired, IGR-NB8-R tumours at passage 8 were further grown without treatment, while the prolonged treatment process was continued in parallel up to 28 passages. Sensitivity to CPT-11 was checked regularly (every 3–4 passages) by evaluating the TGD after one cycle  were grown in athymic mice. The sensitivity of the parental tumour (IGR-NB8) and the resistant tumour (IGR-NB8-R) to other anticancer drugs is shown in Figure 3−1)  containing a high number of mitotic figures per high-power field and very little schwannian stroma , P9 (resistant tumour) and P8P16 (reverted tumour) exhibiting a median value of 26.5, 25, and 20 copies per haploid genome, respectively , respectively. The modifications of topoisomerase I catalytic activity was not significant over these passages, while TGD decreased from 115 to 32 days. Moreover Western blot analysis of topoisomerase I showed no difference between the tumours studied  and at passages 6 (P6) and 11 (P11). Three tumours were studied at each passage. MDR1-mRNA expression was detected in all tumour samples. As compared to GAPDH-mRNA expression, the MDR1-mRNA RER was 1.17±0.46, 1.38±0.43 and 1.23±0.26 at P1, P6 and P11, respectively. These levels of relative expression were comparable with the positive control MCF7DXR 1.44±0.12. MRP1-mRNA was also detected in all tumour samples, with an RER of 2.4±0.05, 1.91±0.36 and 2.08±0.75 at P1, P6 and P11, respectively. These levels of relative expression were comparable to that of A549, the positive control (2.86±0.71). These results confirmed the strong basal expression of MDR1 and MRP1 in IGR-NB8. However, expression of both MDR1 and MRP1 did not increase from P1 to P11, while the TGD was being significantly reduced from 115 to 25 days. Immunohistochemical analysis of BCRP revealed relatively high expression in the positive control (human placenta), and the absence of BCRP expression in sensitive, resistant and chemosensitivity-restored tumours. Northern blotting confirmed these results. BCRP/MXR/ABCP mRNA was undetectable in all the tumours analysed at various passages . The positive control (T8 cells) . Thus, CPT-11 resistance in vivo in IGR-NB8-R xenograft is not related to MDR1, MRP1 and BCRP expression.in vitro studies. Several cell lines selected for resistance to irinotecan have been described and these studies have shed light on mechanisms such as topoisomerase I alteration or increased efflux of irinotecan from the cell  of the EMT-6 mouse mammary tumour through in vivo drug administration to syngeneic BALB/c tumour-bearing mice. The resistance observed was reversible after discontinuation of treatment, as demonstrated in our model. In spite of high levels of resistance in vivo, no significant resistance was observed when the cells from these tumours were exposed to the drugs in vitro, indicating that a very high level of resistance to anticancer drugs can develop through mechanisms that are exclusively in vivo  was observed in the TGD. Third, the number of consecutive cycles that were required for 50% of tumours to attain five-fold their initial volume was reduced from four to two cycles. The CPT-11-resistant IGR-NB8 NB model was designated IGR-NB8-R.Thus, our first objective was to establish a subcutaneous NB xenograft model that is resistant to CPT-11. After a series of 68 cycles of CPT-11 treatment and 28 passages in vivo acquired resistance to CPT-11 has been reported, which is also an in vivo NB (NB-1691) model established by Thompson et al. After four rounds of treatment/transplantation (5 mg kg−1 administration−1), authors observed a resistance to irinotecan and a partial resistance to topotecan. The mechanisms of resistance were not discussed in this paper  activation was shown to play a role in sensitivity to this drug; treatment with camptothecin was reported to activate NFκB (κB. The apoptotic response mediated by CPT-11 was dependent on NFκB inhibition, establishing NFκB as a principal mediator of inducible chemoresistance (κB in our model of acquired resistance to CPT-11.Among the various mechanisms of resistance to irinotecan characterised in vitro preclinical studies. Furthermore, it is a revertible mechanism that probably does not imply mutations. Our next objective is to investigate the basis of this mechanism of acquired drug resistance. Several genes are likely implicated; therefore, a genomic comparison of the transcriptome of sensitive and resistant tumours with macro- and microarray is in progress. This technology will allow us to rapidly identify differences in expression between genes. Clarifying the resistance mechanism in IGR-NB8-R will allow us to make a step forward in our understanding of acquired chemoresistance and to better target the clinical development of irinotecan.We have established a human NB xenograft resistant to CPT-11. This resistance mechanism seems to be novel. It does not imply any of the mechanisms of resistance usually observed in


The effects of thymectomy and sham operation on 9,10-dimethyl-1,2-benzanthracene (DMBA) induced tumours of the hamster cheek pouch were studied in Syrian golden hamsters. The incidence of carcinomata and papillomata with intra-epithelial carcinoma  in these animals was compared with that in control animals treated with DMBA alone, without surgical intervention.In 32 non-operated control animals, the average tumour yield after 12 weeks' DMBA application was 2·13 carcinomata and 1·22 atypical papillomata per treated pouch. In 14 animals thymectomized at the age of 2 weeks, the tumour yield was 0·21 and 0·36, respectively, and in 10 animals thymectomized when adult, it was 0 and 0·1 respectively. In 9 animals sham operated on at the age of 2 weeks, an average of 1·56 carcinomata and 1·22 atypical papillomata were found, but in 13 animals which were sham operated when adult, the tumour yield was 0·54 and 0·15 per treated pouch, respectively.The results suggest that the time of thymectomy in relation to DMBA treatment may be of importance and that thymectomy, when performed in 2-week old hamsters, inhibits DMBA tumourigenesis. The major effect of thymectomy performed in adult hamsters appears to be related to stress following surgery.


Two colorectal  and one gastric (MKN45) human tumour cell lines were examined for their in vitro trophic response to human gastrin-17. MKN45 and HT29 responded by increased 75Se selenomethionine uptake to exogenous gastrin  whereas LoVo showed no significant response to this hormone. When these same cell lines were grown as xenografts in nude mice, similar responses were seen to exogenously administered human gastrin-17 . MKN45 xenografts showed a greater response to continuously administered gastrin  when compared to the same dose given via a subcutaneous bolus injection. The hormone-treated xenografts had a two-fold increase in tumour cross-sectional area and growth rate when compared to saline-treated controls. Dose-response studies revealed that 0.4 micrograms gastrin mouse-1 day-1 appeared to be the minimally effective dose. As gastric and colorectal tumour cells show a trophic response to gastrin, antagonists of the gastrin receptor may prevent this effect causing tumour stasis. The gastric tumour cell line, MKN45, is gastrin-responsive and would be an ideal model for screening potent receptor antagonists.


In the present study, cancer vaccines were prepared by treating mouse SCCVII squamous cell carcinoma cells with photosensitiser chlorin e6-based PDT and used against poorly immunogenic SCCVII tumours growing in syngeneic immunocompetent mice. The vaccine potency increased when cells were post-incubated in culture after PDT treatment for 16 h before they were injected into tumour-bearing mice. Interfering with surface expression of phosphatidylserine (annexin V treatment) and apoptosis (caspase inhibitor treatment) demonstrated that this post-incubation effect is affiliated with the expression of changes associated with vaccine cell death. The cured mice acquired resistance to re-challenge with the same tumour, while the engagement of cytotoxic T lymphocytes was demonstrated by detection of high numbers of degranulating CD8+ cells in vaccinated tumours. The vaccines prepared from ex vivo PDT-treated SCCVII tumour tissue were also highly effective, implying that surgically removed tumour tissue can be directly used for PDT vaccines. This opens attractive prospects for employing PDT vaccines tailored for individual patients targeting specific antigens of the patient's tumour.Recent investigations have established that tumour cells treated  Although photodynamic therapy (PDT) is established for clinical treatment of neoplastic and other lesions , this moin vitro by PDT, was shown to effectively protect against challenge of mice with the same tumour cells. The authors showed that the action of this prophylactic vaccine is associated with phenotypic and functional maturation of dendritic cells and the induction of a cytotoxic T-cell response, and is based on tumour-specific immune response (ineffective against mismatched tumour types). Therapeutic PDT vaccine preparations based on whole tumour cells were developed in our laboratory  and include the necessary determinants for helper T cells . Autologin vivo by serial transplantation into syngeneic C3H/HeN mice. Experimental tumours were initiated by injecting subcutaneously 1 million cells in the lower dorsal region of 7–9 weeks old female mice. The cultures of SCCVII cells in vitro were grown in Alpha Minimal Essential Medium  supplemented with 10% fetal bovine serum . The vaccination was carried out as a single peritumoral injection (unless stated differently), and was performed at 6 days post-tumour implant when these lesions reached 5 mm in largest diameter. After vaccination, tumour sizes were monitored by measuring the lesion's three orthogonal diameters with a caliper. The follow-up period after vaccination lasted until the tumours in control groups became too large (>12 mm largest diameter) for keeping the mice due to ethical considerations. In cases of vaccine-induced complete tumour regression, the mice were kept under observation for up to 90 days and those remaining tumour-free at that time were declared cured. Each treatment group consisted of six mice. The protocols used with mice were approved by the Animal Care Committee of the University of British Columbia.Mouse squamous cell carcinoma SCCVII, recognised as an excellent model for poorly immunogenic head and neck cancer of spontaneous origin (μg ml−1) in serum-free medium for 30 min at 37°C, washed and exposed to 1 J cm−2 (15 mW cm−2) of 665±10 nm light, then treated with X-rays (60 Gy), and immediately thereafter injected peritumorally . The light source used was an FB-QTH-3 high throughput illuminator  equipped with a 150 W QTH lamp and suitable interference filter. The X-ray source was Philips RT 250 . Control groups included tumour-bearing mice injected with the same number of SCCVII cells that received only X-ray treatment (no PDT).In initial experiments, vaccine generation procedure described in a previous report  was foll7 cells per 175 cm2 T-flask with 20 ml medium. The cells were then concentrated for X-ray treatment and injection into mice. The medium in which the cells were ‘post-incubated’ was in some cases also collected, then concentrated using molecular filters , and included together with the vaccine cells into the 0.2-ml volume injected into mice.In the course of this study a new protocol was developed referred to as ‘post-incubation vaccine protocol’. It differs from the standard vaccine protocol only in including a 16-h (overnight) incubation of SCCVII cells at 37°C after they were treated with PDT light; the cells were kept in a specially enriched serum- and protein-free medium  distributed at 4 × 10μg ml−1 (50 μM) at the onset of 16-h post-incubation of PDT-treated SCCVII cells. Alternatively, the cells were post-incubated without Z-DEVD but were exposed immediately thereafter to annexin V  at the concentration of 0.2 mg ml−1 in annexin V binding buffer  for 30 min at room temperature. The cells were then collected, concentrated for X-ray treatment and injected as PDT vaccine.To investigate the role of apoptotic cell death in PDT vaccine cells, apoptosis blocking agent caspase-3 inhibitor benzyloxycarbonyl-Asp(OMe)-Glu(Ome)-Val-Asp(OMe)-fluoromethyl ketone  was added to the medium at the concentration of 33.4 ex vivo with ce6 (0.5 μg ml−1) and further processed according to the post-incubation vaccine protocol. The vaccine injection volume (200 μl) contained 170 μl of wet brei plus 30 μl of phosphate-buffered saline (the highest brei concentration that can pass through the needle).For preparing the PDT vaccine from tumour brei, tissue of growing untreated tumours was finely minced using a pair of scalpels and frozen until use in liquid nitrogen. Freshly thawed brei was incubated 2a (eBioscience). The samples were analysed with a Coulter Epics Elite ESP  including 20 000 cells for each test. Degranulating cytotoxic T lymphocytes were identified as CD8+CD107a+ cells from populations gated negative for myeloid marker Mac-1.The procedure used was recently described in detail . BrieflyAdditional flow cytometry analysis was carried out for determining the percentage of apoptotic and necrotic cells that appear following the post-incubation protocol for PDT vaccine generation. For this purpose the cells were stained with FITC-conjugated annexin V followed by PE-conjugated rabbit monoclonal antibody against active caspase-3 diluted in Perm/Wash buffer . Apoptotic cells were identified as positively stained with both antibodies, while necrotic cells were positively stained only with annexin V.Tumour growth-inhibition results and mouse survival results were statistically evaluated using log-rank test. The differences between means of the data from remaining results were analysed with Mann–Whitney test. Significance level of 5% was set as a threshold for determining if the groups were statistically different.in vitro expanded SCCVII cells to either 0.5 or 2.5 μg ml−1 ce6 for 30 min in serum-free medium followed by treatment with 1 J cm−2 of 665±10 nm light. The PDT dose with the lower ce6 concentration kills around ⅔ of cells, while treatment involving the higher ce6 concentration is supralethal (data not shown). Immediately thereafter, the cells were concentrated, treated with a lethal dose of X-rays and delivered by perilesional injection (2 × 107 per mouse) to mice-bearing established SCCVII tumours. Except for the photosensitiser, this procedure followed the protocol optimised in our previous studies . This was manifested as an initial growth-inhibitory effect including most or all tumours and persisting in the one-third of tumours with the PDT vaccine group based on the lower ce6 dose (0.5 μg ml−1) that appeared more effective than the PDT vaccine using the higher ce6 dose . In this experiment, the tumours that qualified as growth-inhibited at 9 days post-vaccination become impalpable and showed no signs of recurrence up to 90 days post-vaccination which categorises them as cured. On day 100 post-vaccination, these cured mice were re-challenged by an inoculum of 1 × 106 SCCVII cells. Subsequent monitoring showed no tumour growth, which demonstrates that the mice acquired resistance against the vaccinated tumour.The effectiveness of PDT vaccines prepared by employing photosensitiser ce6 (not tested before for this application) against subcutaneous SCCVII tumours growing in C3H/HeN mice is shown in  studies . Also in studies . Statistμg ml−1 was chosen for PDT vaccines in the remaining experiments of this study. Examined next was the possibility that the effectiveness of PDT vaccine can be augmented by keeping the vaccine cells after PDT treatment under culture conditions at 37°C to allow the progression of induced molecular/biological events that may prove relevant for the vaccine's action. Hence, tumour-bearing mice received in the next experiment either the standard PDT vaccine (cells used immediately after PDT treatment) or PDT vaccine consisting of cells incubated after PDT treatment for 16 h at growth conditions before harvested for injection (post-incubation vaccine protocol). The response  is shown in The ce6 dose of 0.5 In the next experiment, one group of tumour-bearing mice received the vaccine that contained not only PDT-treated cells collected after the post-incubation protocol but also the concentrated supernatant (medium) collected from the same cultures. At 5, 7 and 9 days post-vaccination the tumours in this group were significantly smaller than the tumours in unvaccinated control group but their size was not significantly different than the size of tumours in the group that received the PDT-treated and post-incubated cells without the supernatant . On the P<0.05), this effect was almost completely abolished in mice receiving annexin V modified vaccine (Exposure of PDT vaccine cells after the 16-h post-incubation to annexin V (known to bind phosphatidylserine) dramatically affected the efficacy of the vaccine. While PDT vaccine not involving annexin V exposure strongly inhibited tumour growth , but the difference compared to the unvaccinated controls was not statistically significant.The results of previous investigations of PDT vaccines suggest that their effect is based on the action of tumour-specific cytotoxic T lymphocytes . To obtaTo test the relevance of the distance of vaccination site from the targeted tumour, the effects of PDT vaccine delivered locally  and at a distant site  were compared in the same experiment. The results, showing the percentage of growth-inhibited tumours during the post-vaccination follow-up, are depicted in ex vivo to ce6-based PDT followed by overnight (16-h) post-incubation. The samples were then prepared for vaccination by insuring that each injection contains 170 μl of wet brei; this was treated with X-rays (60 Gy) and injected peritumorally. The results show that the brei-based PDT vaccine was very effective in inhibiting tumour growth and exhibited potency close to that of cell-based PDT vaccine tested in the same experiment  of SCCVII tumours (freshly thawed from storage in liquid nitrogen) instead of SCCVII cells was exposed periment .in vitro by PDT can be used for generating potent cancer vaccines (+ cells were found in lesions regressing after PDT vaccine treatment and much lower numbers in the tumours within the same treatment group that exhibited a poor early responsive (progressing after vaccination). Elevated numbers of degranulating CD8+ cells were also found in tumours treated by a regular (direct) PDT treatment , the proximity of vaccination site to the treated lesion is relevant for the therapy outcome. While PDT vaccines injected at a distal site were still effective, their impact was inferior to perilesional treatment. This is probably due to the fact that antigen presentation to T cells takes place in the lymph nodes nearest to the vaccine injection site. In case of peritumoral vaccine injection T-cell activation will be centered in tumour-draining lymph nodes, which is a location favourable for T-cell trafficking into the tumour. Thus with the proximal treatment the vaccine effect could be expressed more rapidly than with the distal. Such delay in PDT vaccine-mediated immune activity may prove less important in tumours with slow growth rate.In our previous studies, we have identified several key parameters for optimal action of PDT vaccine such as the number of PDT-treated tumour cells to be used per vaccination and the concentration for photosensitisers that were employed  . In the This study demonstrates that the potency of PDT vaccine is increased when vaccine cells remain in culture after PDT treatment for an additional time interval (post-incubation protocol) to allow the expression of PDT-induced molecular/biological changes in these cells. Such changes of possible relevance include the progression of apoptotic or necrotic death process associated with the appearance of death signal molecules on the cell surface . The exp3 at the time of vaccination. Obviously, PDT vaccines are more effective in eradicating smaller tumour mass but this does not mean that they cannot be employed for therapy of larger lesions. Decreasing the tumour burden by radiotherapy before PDT vaccine treatment allows curing mice with relatively large malignant growth. For instance, the treatment of SCCVII tumours by 20 Gy produced no permanent cures but if this X-ray dose was followed by PDT vaccine application  it resulted in 50% tumour cures . We are also confident that the potency of PDT vaccines can be further increased by adjuvant immunomodulatory treatment.Tumour responses to PDT vaccine showed some variation in the intensity in different experiments. However, in our continuing studies we are now routinely achieving the cures of the two-thirds of PDT vaccine treated tumours; this appears to result from insuring that no tumours are larger than 30 mmThis study also demonstrates that tumour tissue can be directly used for the production of PDT vaccine without the need for generating first cultures of single cancer cells. This has very important clinical implications, since tumour tissue surgically removed from the patient can be used for preparing without delay (and with avoiding difficulties/uncertainties in establishing primary cancer cell culture) the PDT vaccine material directly tailored for the individual patient which is acting against tumour antigens existing in that specific tumour.


To improve the efficiency of hepatic intra-arterial (h.i.a.) chemotherapy, we selected pirarubicin (THP) because it shows good properties for h.i.a. chemotherapy, such as fast and efficient cellular uptake, and used it for h.i.a. chemotherapy in rabbits with V x 2 tumour implanted in the liver. The anti-tumour effect of THP upon h.i.a. administration was compared with that upon intravenous (i.v.) injection and also with the anti-tumour activity of epirubicin (EPI) upon h.i.a. injection using optimal and maximal tolerated doses of each drug. When tumour growth rates and morphometric examinations were evaluated, it was found that THP and EPI were effective against V x 2 tumour when injected via the h.i.a. route. The activity of THP was stronger than that of EPI. As regards h.i.a. injection-related complication, plasma transaminase levels were temporarily elevated. To demonstrate higher anti-tumour activity and other advantages of h.i.a. injection of THP, plasma and tumour drug concentrations were determined by high-performance liquid chromatography after THP or EPI was administered at an equal dose to the rabbit V x 2 model. Hepatic intra-arterial injection of THP accomplished a selective and higher uptake into the tumour and lower effusion into the plasma than i.v. injection of THP or h.i.a. injection of EPL. Our findings indicate that THP is the better candidate of the two drugs tested for the h.i.a. chemotherapy because of its greater anti-tumour activity and the lower systemic drug exposure achieved upon h.i.a. injection.


The majority of human anti-tumour monoclonal antibodies (Mabs) isolated to date have been disappointing. Firstly, they react or cross react with intracellular cytoskeletal proteins or nuclear antigens and therefore are of limited value as blood borne agents. They are also generally of the IgM isotype and show relatively low intrinsic affinity for the primary epitope. Secondly, such Mabs can be generated from normal, non tumour bearing subjects at a frequency comparable to their production from tumour patients. This latter observation is true also for common autoantigens such as DNA and IgG since Mabs to these can also be generated from normal subjects in addition to autoimmune individuals. This article rationalises these observations in the context of the requirement for clinical use for human Mabs. It discusses the evidence that there is a potentially useful B cell response to be immortalised, and examines the consequences of the newly recognised phenomenon of monoclonal antibody multispecificity both on the methodology of their generation and on their subsequent use as imaging and therapeutic tools.


Carrageenan (Cg) was tested for its effects on the growth of, and immunity to, 2 methylcholanthrene-induced syngeneic murine fibrosarcomas (H1 and H2). The tumours were found not to share major tumour-specific transplantation antigens. H2 appeared more immunogenic than H1. In contrast to H1, immunity induced by H2 was not affected by Cg, nor was its growth in Cg-treated normal mice augmented.immune mice was significantly greater than the augmented growth in Cg-treated normal mice. The Prior administration of the macrophage-stabilizing agent polyvinylpyrrolidone (PVP) to immune mice significantly reduced the augmenting effect of Cg. The growth-promoting effect of Cg on a secondary H1 tumour challenge in mice immunized by tumour excision was abolished by 106 MCT-H1 cells injected s.c. before Cg. In contrast to the immunity induced by tumour excision, Cg did not abolish the immunity induced by the injection of MCT-H1 cells.Postoperative i.p. injections of Cg abolished the weak anti-H1 immunity produced by H1 tumour excision. Furthermore, the subsequent growth of the H1 tumour challenge in the Cg-treated in vivo by MCT-H1 cells. However, TBS administered to Cg-treated, MCT-H1-immune mice abolished tumour immunity.Passive administration of H1 tumour-bearer serum (TBS) did not enhance the growth of H1 cells in normal mice, nor did TBS abrogate the specific cell-mediated immunity (CMI) induced in vivo provided that macrophages remain functional, but may do so when macrophages are rendered defective by antimacrophage agents or by products of neoplastic cells. Increasing the levels of specific effector cells can over-ride the inhibiting effects of TBS, even when defective macrophages are present.We propose that TBS does not inhibit CMI


Reactivity of lymphocytes from lymph nodes, spleen and blood was tested throughout the period of tumour growth. Maximum lymphocyte cytotoxicity against the tumour was observed at 2 weeks in regional lymph nodes, 4 weeks in intermediate nodes, spleen and blood, and 6 weeks in distant nodes; the intensity of these cytotoxic responses subsequently declined. In the regional nodes, lymphocytes became totally unresponsive despite the maintenance of significant cytotoxicity in intermediate nodes, spleen and blood. Local anergy may account for tumour spread to the regional node in an otherwise immunocompetent host. This anergy may be due to high local concentration of tumour antigen or antigen-antibody complexes, but it was not associated with selective changes in T and B cell proportions.The development of cellular immunity to a syngeneic squamous cell carcinoma in Wistar rats was studied by


The combined effect of the chemotherapeutic agent ICRF-159 and irradiation were evaluated using the Lewis lung tumour (LL). At a daily dose of 25 mg/kg, ICOF given alone prevented the progressive growth of LL. Daily pretreatment also potentiated the effects of radiation (600 rad) on tumour growth, provided the pretreatment kinetics of the tumour permitted a response to radiation alone. Single acute doses of the drug failed to alter the growth of LL, and when combined with radiation failed to enhance the radiation effect. Fractionation of the drug  before irradiation, however, results in immediate effects on tumour growth which are more than additive. The results suggest that a low dose of ICRF-159 for extended periods is more effective in enhancing radiotherapy than a high dose provided acutely.


Most of the modeling modules have been based on discrete mathematics and therefore have been formulated in terms of rather complex algorithms . However, such lengthy algorithmic descriptions, although sufficient from the mathematical point of view, may render it difficult for an interested reader to readily identify the sequence of the very basic simulation operations that lie at the heart of the entire model. In order to both alleviate this problem and at the same time provide a bridge to symbolic mathematics, we propose the introduction of the notion of hypermatrix in conjunction with that of a discrete operator into the already developed models. Using a radiotherapy response simulation example we demonstrate how the entire model can be considered as the sequential application of a number of discrete operators to a hypermatrix corresponding to the dynamics of the anatomic area of interest. Subsequently, we investigate the operators’ commutativity and outline the “summarize and jump” strategy aiming at efficiently and realistically address multilevel biological problems such as cancer. In order to clarify the actual effect of the composite discrete operator we present further simulation results which are in agreement with the outcome of the clinical study RTOG 83–02, thus strengthening the reliability of the model developed.The tremendous rate of accumulation of experimental and clinical knowledge pertaining to cancer dictates the development of a theoretical framework for the meaningful integration of such knowledge at all levels of biocomplexity. In this context our research group has developed and partly validated a number of spatiotemporal simulation models of  The usually fast growth and resilience of tumours suggest that they are emerging, opportunistic systems rather than random, disorganized and diffuse cell masses.in vivo growing tumour rather than only a single cell4An inelastic prerequisite for an effective treatment of cancer is understanding and modeling the corresponding spatiotemporal natural phenomenon of tumour growth and response to therapeutic schemes concurrently on several in vitro case or the early avascular stages of small in vivo tumours and is based on a consideration of the distinct phases of the cell cycle. Kocher et al1, S, G2, M) had not been taken into account, grouping of the cells into only proliferating and dormant classes being considered instead. It is noted that none of the above mentioned models has been applied to large clinical tumours  and none of them simulates shrinkage for an arbitrarily shaped clinical tumour undergoing treatment. In the pure tumour growth models presented by Kansal et alin silico (=on the computer) has also been presented by Deisboeck et al.16This section provides a brief outline of several of the concepts and earlier research efforts to model tumour behaviour. Duechting et alin vivo simulation models of imageable tumour response to radiotherapeutic and chemotherapeutic schemes.An effort to overcome some of the above mentioned limitations has been previously made by our research group through the development of four‐dimensional patient-specific in silico experiments. At the same time as cancer is an excellent paradigm of multilevel biological phenomena any rigorous theoretical treatment of cancer dynamics could provide important hints for the modeling and simulation of other biological phenomena including other homeostatic imbalances (diseases).natural phenomenon of cancer are incorporated into mathematical and computational models important discrete mathematics modeling treatments tend to become difficult to understand by the wider cancer modeling community. Therefore, the need for a symbolic mathematical notation has become obvious. Such an approach could be viewed as following the formalist school founded by D. Hilbert.formal symbolic systems.Looking now at the problem of cancer from a broader theoretical perspective we realize that the impressive rate of production of experimental and clinical knowledge pertaining to the disease dictates the development of a generic and desirably universal theoretical framework for the meaningful integration of such knowledge. The obvious reasons for knowledge integration are both deeper understanding of cancer and optimization of therapeutic schemes on the patient individualized context by performing in vivo making use of several versions of the simulation model developed by our group (see citations above). In order to proceed to the introduction of a discrete mathematics operator notation the anatomic region of interest and its biological dynamics are represented by a hypermatrix ᾱ. A hypermatrix can be viewed as a matrix of [a matrix of [… of [matrices (or vectors) ]…]]. The hypermatrix ᾱ is created by the superposition of a discretization mesh on the anatomic region of interest and the consideration of equivalence classes within each geometrical cell of the mesh representing the various phases within or out of the cell cycle that a biological cell can be found. Discrete time represents a further dimension of the hypermatrix.Stimulated by these remarks we present the introduction of an operator notation into the malignant tumour response to therapeutic schemes in vivo discrete mathematics treatment of the tumour growth and therapy response phenomenon. A discretizing mesh is superimposed on the anatomic region of interest and the contents of each geometrical cell of the mesh are distributed into equivalence classes corresponding to the various phases within or out of cell cycle. The mean time spent within each phase is another parameter that characterizes the subsets of each geometrical cell.24Collection of the appropriate imaging data , registration, interpolation and three dimensional reconstruction constitute the initial steps of an ā stands for the hyper‐matrix corresponding to and dynamically describing the anatomical region of interest . Each vector element of the hypermatrix ᾱ is considered to have the following form that corresponds to a discretization mesh geometrical cell:The following mathematical entities are considered in the proposed treatment: where the following symbols have been introduced:p: phase within or out of the cell cycleg: oxygen and nutrient provisionNp: number of biological cells in phase ptp: mean time spent in phase p hp: number of therapy hit cells residing in phase ph̃p number of non therapy hit cells residing in phase pwhereξmin, ξmax denotes the minimum and maximum value respectively of the generic variable ξ during the simulationG1 denote the G1 cell cycle phase,S denotes the DNA synthesis phase,G2 denotes the G2 cell cycle phase,M denotes mitosis,G0 denotes the dormant G0 phase,A denotes the apoptotic phase,N denotes the necrotic phase,D denotes the remnants of dead cells,s stands for suffcient oxygen and nutrient provision (for tumour cell proliferation), s̃ stands for insuffcient oxygen and nutrient provision (for tumour cell proliferation).Obviously this binary character of the oxygen and nutrient provision is to be considered only a first simplifying approximation.N0 is the set of non negative integers0 phase located around a given point may imply inadequate oxygen and nutrient provision, although in this particular case dormancy is normally the outcome rather than the cause of inadequate oxygen and nutrient provisionIt should be noted that in general all physicobiologically different components of each hypermatrix element can be considered dependent on all five dimensions of the proposed abstract space of tumour dynamics. For example oxygen and nutrient provision can change dramatically in space and time within the tumour. The treatment outcome is generally dependent on the phase in which a cell resides when irradiated or treated with chemotherapy. Cell cycle phases have generally different durations and therefore the mean time spent within each phase equivalence class of a given geometrical cell is dependent on the cell phase. Even the oxygen and nutrient provision  may be microscopically related to the phase under consideration. A relatively large number of tumour cells within the Gxi, yj, zk) represent space, another one (corresponding to the variable tn) time and the fifth one (corresponding to the variable pl) represents the cell phase within or out of the cell cycle in which a biological cell or a set of cells within a geometrical cell of the discretization mesh is found at a given instant. The entire simulation can be viewed as the periodic application of a number of discrete algorithmic operators on the hypermatrix of the anatomic region of interest. The period of application is equal to the time separating two consecutive discretization mesh scans. This has been taken equal to 1h in all applications referred to in this particular paper.The various modules of algorithmic manipulations on the hypermatrix can be thought of as corresponding to discrete operators acting on the hypermatrix in analogy to the action of continuous operators on a wave function in quantum mechanics .O stands for the oxygen and nutrient provision statusH stands for the effect of therapy C stands for the eventually perturbed cell cycling due to therapyE stands for differential expansion or shrinkageU stands for oxygen and nutrients field updateor in a more compact writing:where the application of the operators takes place from the right to the left.partial operator as used in this work essentially denotes the application of complex algorithmic manipulations. The entire model has been constructed with a number of algorithmic manipulations  applied repeatedly in a given order. Therefore, the whole model  can be considered as the application of a composite operator. Subsequently this composite operator can always be decomposed into partial operators since the former is nothing more than a conceptual clustering of the latter.It is noted that the term It is quite obvious that the above mentioned concepts and symbols cannot include all the information needed for the simulation to run. Their role is (at least at the present stage) rather to identify and decompose the major conceptual mathematical treatment steps than to represent any assumption details. The proposed approach is to be seen as a continually evolving and optimized process. Examples of such evolutionary stages could be the following. In order to address the non imageable components of highly invasive tumours such as glioblastoma multiforme at a large time scale further operators could be proposed so as to explicitly handle diffusion phenomena at the cellular/tissue level. Additional operators could handle the refined biomechanics of the tumour and adjacent normal tissues as well complex molecular networks which largely determine the response of a single tumour cell to treatment (see Section 5).fJ, fK where J, K ∈ {U, E, C, H, O, T } reveals that they are non commutative. It is noted that the terms commutative and permutable are used interchangeably when applied to operators .fC) to the proliferating cells first and subsequently calculating the effect of a radiotherapy fraction (fH) may lead to a substantially larger number of surviving cells than the opposite sequence. The reason for such a difference could be the successful completion of mitosis for far more tumor cells in the first case which would lead to a larger number of tumour cells (tumour burden) before treatment is applied. Furthermore, it must be stressed that in general each partial operator has to be applied concurrently on all elements‐geometrical cells of the hypermatrix ᾱ  as there are in general interdependences among the various geometrical cells [e.g. differential shifting of the overflowing that have emerged following mitoses].A careful study of the behaviour of any two of the “partial operators” f to its constituent “partial operators” fJ, J ∈{U, E, C, H, O, T} provides a conceptual tool useful for the analysis and superposition of several critical mechanisms that may take place on different biocomplexity levels. For example the molecular profile of a given tumour  can be used in order to perturb the population based average survival fraction following irradiation with dose D so that the genetically produced radiosensitivity or radioresistance of the particular tumour of a given patient is taken into account during the simulation. This “summary” of the molecular level phenomena is currently incorporated into the partial operator fH which represents the microscopic effect of the treatment intervention on the tumour. One could interpret the procedure of perturbing the population based average values of the tumour biological parameters as a two step process. The first step refers to “summarizing” what is happening on one biocomplexity level (here the molecular level) and providing the amount of perturbation whereas the second one refers to the “jumping” to another level (here the cellular level). In this case the “summary” refers to the percentage by which the survival fraction will have to be perturbed whereas the “jumping” refers to the fact that the individualized survival fraction is related to the cellular level on which the main bulk of the simulation process takes place. Furthermore, it is worth noting that it is at the cellular level that complete success or failure of tumour treatment is defined since complete success of tumour treatment implies that not even a single proliferative or dormant tumour cell has been left alive.In order to achieve a quite realistic prediction of the response of a tumour to therapeutic interventions several levels of biocomplexity have to be addressed at the same time. The decomposition of the composite discrete operator T1 gadolinium enhanced) imageable glioblastoma response to radiotherapeutic schemes. As a thorough account of the assumptions made would be beyond the scope of the present paper we only provide an indication of the operator notation introduction by referring to selected modeling points. Further modeling details can be found i.a. in.27The introduction of the suggested notation is demonstrated through a version of a simulation model of  p53 function, and accordingly adjusted LQ Model parameters.35A GBM tumour with mutant (mt) p53 geneA GBM tumour with intermediately adjusted radiosensitivity:In all cases, we set Kocher; p. 99:andThe meaning of the symbols used is the following:αP, βp: the LQ Model parameters for all proliferative cell cycle phases except for the DNA synthesis phase (S phase).αS, βS: the LQ Model parameters for the S phase.αG0, βG0: the LQ Model parameters for the resting G0 phase.The delivery of irradiation takes place at 08:00 and 16:00 every day, 5 days per week (no irradiation during weekends). The distribution of the absorbed dose in the tumour region is assumed to be uniform. It should also be noted that carmustin, which was administered to all patients enrolled in the RTOG—83-02 study, is assumed not to significantly modify the relative effectiveness of the radiation therapy schedules considered, as the chemotherapy administration schedule was the same for all patients.The first process that takes place before the application of the operators is the discretization of the anatomic region of interest and the construction of the corresponding hypermatrix. The imaging data  including the definition of the tumour contour, its metabolically active sub‐regions and the anatomical structures of interest, the histopathological (e.g. type of tumour) and the genetic data (e.g. p53 status and other molecular data) of the patient are collected. The clinician delineates the tumour and the anatomical structures of interest by using a dedicated computer tool. In the case of radiotherapy, the planned distribution of the absorbed dose (e.g. in Gy) in the region of interest is also acquired. For the purpose of the 3D reconstruction and visualization of both the initial tumour and the simulation outcome, the 3D visualization package AVS/Express 4.2 has been used, .4 to 105 cells/mm3.5 cells/mm3 in the proliferating cell region, 105 cells/mm3 in the G0 cell region and 0.2 × 105 cells/mm3 in the dead cell region of the tumour.We assume that each GC of the mesh initially and normally accommodates a Number of Biological Cells (NBC). However, the maximum number of biological cells that is allowed to be accommodated within a GC is assumed to be NBC + [a fraction of NBC]. NBC apparently depends on the selected size of the GC and determines the quantization error of the model. The fraction of NBC considered in the code executions in this paper was 1/10 as this was shown to be the optimal one from both the convergence and CPU time demands points of view.It is noted that each multidimensional element of the proposed hypermatrix has a clear physical and/or biological meaning . Therefore, the hypermatrix (such as the exemplary one outlined above) is used to describe the distribution of several critical physical and biological quantities over space and time.In the following the various processes constituting the entire simulation algorithm are briefly and separately described with reference to the corresponding operators.Time is discretized and incremented. One hour has been adopted as the unit of time since 1h is the approximate duration of mitosis, the shortest cell cycle phase. According to the prescribed radiotherapy scheme the specific instants corresponding to the delivery of a radiation dose to the tumour region are defined. In each time step the geometrical mesh is scanned and the updated state of a given GC is determined on the basis of a number of behaviour algorithms:1, S, G2, Mitosis) is considered based on experimental evidence (Katzung (Ed),During each scan of the discretization mesh the effect of the oxygen and nutrient provision on the cells of each geometrical cell is taken into account. This provision, determining the metabolic potential of a region, is based on the imaging data and determines the distribution of the tumour cells in the proliferative, dormant and necrotic states within the regions without taking into account the eventual therapeutic interventions effects. Furthermore, distribution of the cells over the cell cycle phases (G(I) At the time instants corresponding to the delivery of a specific radiation dose to the tumour (according to the prescribed radiotherapy scheme and the acquired distribution of the absorbed dose in the region of interest) the number of cells killed in a particular GC is calculated based on the Linear Quadratic (LQ) Model, which is widely used in the pertinent literature.−1) and β (Gy−2) characterize the initial slope and the curvature, respectively, of the survival curve.where α  Lethally damaged cells following exposure to radiation undergo two mitotic divisions prior to death and disappearance from the tumour.39Note: Any eventual molecular perturbators of the cell surviving fraction are to be incorporated into this operator.1→S , S→G2 , G2→M , M→G1 or M→G0 , G0→G1 (if adequate oxygen and nutrient provision has been re‐established) or G0→N . The previously mentioned durations of the cell cycle phases TX, X ∈ {G1, S, G2, M, G0} seem to follow the normal distribution according to pertinent literature. As a first approximation, we use the mean values of the duration of each cell cycle phase and neglect standard deviations. However pseudo‐random number generators are used in order to de‐synchronize the equivalence classes throughout the tumour.At each time step the time registers of all GCs are incremented by one hour. Cell loss due to apoptosis and necrosis is computed. According to the cytokinetic model appearing inC has been taken equal to 40 h. This is the average of the cell cycle durations we have found in the literature for GBM cell lines.1 = 0.4TC, TS = 0.39TC, TG2 = 0.19TC, TM = 0.02TC. The duration of the G0 phase is taken to be TG0=25 h.42The cell cycle duration TThe cell loss factor (CLF) is considered equal to 0.3.44The differential tumour expansion and shrinkage algorithms are based on the use of random number generators in conjunction with adequately formed morphological rules. These rules lead to tumour shrinkage or expansion conformal to the initial shape of the tumour . Two versions of the expansion and shrinkage algorithms have been tested. First version (a): For each GC, one out of the six possible directions of shrinkage or expansion is randomly chosen (Cartesian coordinate system XYZ centered at the current GC. Each axis defines two possible directions of movement). Second version (b1) Shrinkage: The outermost tumour GC is detected along each one of the six possible directions of shrinkage (Cartesian coordinate system XYZ centered at the current GC. Each axis defines two possible directions of movement). Its “6‐Neighbour” GCs belonging to the Tumour (NGCT) are counted. The direction corresponding to the maximum NGCT is finally selected out of the six possible directions as the direction along which the shifting of the GCs will take place (shifting direction). In case that more than one shifting directions have the same maximum NGCT, then the selection is based on the use of a random number generator. Second version (b2) Expansion: A similar, though inverse, morphological—mechanical rule can be applied in the case of tumour expansion. The need for the formulation of the second improved version of the tumour shrinkage and expansion algorithm has arisen from the inspection of the macroscopic results of the simulation algorithms. Specifically, the completely random selection of one out of the six possible shifting directions, according to the first version, results in a premature extensive fragmentation of the tumour region in case of radiotherapy, which is usually incompatible with clinical experience. The general trend is a conformal shrinking of most solid tumours  as a function of time, for the tumours with mutant p53, wild‐type p53 and the tumour with intermediate radiosensitivity, correspondingly. Improved tumour control following high‐dose HF irradiation is evident in the diagrams and is in agreement with the conclusions of the clinical study RTOG 83‐02. In fact, the higher the total dose in an HF schedule, the better the result in terms of tumour cell kill. It should be noted that the clinical study compared the treatments based on patient survival whereas the simulation results presented refer to tumour responsiveness. Although a correspondence of patient survival with tumour responsiveness may not always hold on a single patient basis, when taking into account relatively large clinical trial populations such a correspondence becomes much more reasonable.In order to clarify the actual effect of the composite discrete operator More specifically, the inspection of the simulation results reveals that AHF schedules, which employ a higher fraction dose compared to HF schedules, seem at first to be beneficial as they achieve the maximum tumour cell kill at some instant. Nevertheless, the duration of the AHF schedules is smaller; as a result, if they fail in eradicating “all” tumour cells, tumour repopulation begins earlier (as in the cases of the tumour with mt p53 and the tumour with intermediate radiosensitivity). Only in the case of the tumour with wt p53, do all radio‐therapy schemes kill all the clonogenic cells we have initially assumed ), so the tumour does not regrow after the end of the treatment. Of course, regions of potential microscopic disease have not been considered, and the accuracy of the simulation model decreases as the number of tumour cells is reduced.symbolic mathematical expressions. Such expressions could by themselves provide hints for further questions, investigations and optimizations due to their inherent logical and quantitative associations. Especially the analogy of the operator notation in biomedicine with their use in several fields of physics such as classical and quantum mechanics can act as a source of guidance and quantitative insight into the hypercomplex biological phenomena.The introduction of operator notation into the process of modeling malignant tumour response to therapeutic schemes has led to a brief and comprehensive description of the major steps of the simulation process. In this way highly complex algorithmic treatments are decomposed into simpler procedures which are readily identifiable by the wider research community. The use of mathematical symbols to denote complex algorithmic processes is expected to function as a stimulant for the advancement of multilevel biological modeling through in silico oncology action of the European Commission (EC) funded project “ACGT: Advancing Clinicogenomic Trials on Cancer” (FP6‐2005‐IST‐026996). In particular the development of the software simulation tool named “Oncosimulator”Obviously the treatment presented should be viewed only as an initial step of a rather long term modeling process as more and more experimental and clinical knowledge could be incorporated into the models which are under continuous development and optimization. Specific aspects that are currently addressed in parallel include i.a. the mitotic potential categories of the cancer stem, progenitor and differentiated cells (leading to a new dimension in the discrete abstract space),The introduction of the proposed hypermatrix and operator notation in order to denote, decompose and identify complex biological mechanisms that contribute to the hypercomplex phenomenon of malignant tumour growth and response to therapeutic schemes has been presented through the use of a discrete mathematics simulation model concerning radiotherapy response. Several aspects of the model had been developed by our research group in the past. Symbolic operator notation has provided a compact way of describing the most crucial simulation steps thus offering a possible basis for quantitative multilevel biology based on discrete mathematics. Furthermore, simulation results mimicking branches of the RTOG 83‐02 clinical study have provided both clarification of the actual content of the composite discrete operator and additional evidence for the potential of the simulation approach presented.


Decreased membrane rigidity is one of the characteristics of malignant cells, resulting in part from the desaturation of stearic acid into oleic acid. In this study we investigated the influence of stearic acid on tumour cell inhibition in vitro and tumour development in vivo. Stearic acid inhibited the colony-forming ability of 4 out of 5 rat and two human tumour continuous cell lines in vitro. In contrast, the colony-forming ability of rat fibroblasts was not inhibited and that of human foetal lung fibroblasts was inhibited at a higher dose than that required to inhibit human tumour cell lines. Using a model of rat mammary carcinoma induced by nitroso-methyl urea (NMU) the subcutaneous injection of stearic acid at weekly intervals prevented tumour development in 5 to 10 rats. Using iodostearic acid twice weekly, 11 of 19 rats were alive and tumour free at week 22 whilst all of 14 animals injected with NMU alone had died of tumour by the 16th week. The ratio of stearic to oleic acids in erythrocyte membranes was significantly reduced in the tumour-bearing rats, but was normal in tumour-free animals treated with stearic or iodostearic acid. These preliminary data indicate that stearic acid inhibits tumour development in rats.


A phenomenon by which pre-irradiation of the abdomen of mice reduced the lung-colony-forming efficiency of i.v.-injected tumour cells is described. The extent of lung-colony inhibition was shown to depend on both the dose and timing of abdominal irradiation. The maximum inhibitory effect was obtained when mice received 1200 rad gamma-irradiation to the abdomen 5--7 days before tumour-cell challenge, but there was no effect when abdominal irradiation was given 1 or greater than or equal to 14 days before challenge, or when radiation doses were less than 600 rad. In mice less than 3 weeks old, the effect was much less marked than in adults. The target tissue which, when irradiated, exerted the inhibitory influence on lung-colony formation was located in the ventral half of the abdomen in all 4 quadrants, and was probably gut. Radioactively labelled tumour cells were arrested normally in the lungs of irradiated mice, but were cleared more rapidly without evidence of sequestration in the irradiated gut. The most plausible mechanism seems to be that irradiation of the gut induces the production of natrual killer cells with anti-tumour activity, though this has not been conclusively established.


HPMA copolymer-cathespin B exhibited a longer plasma half-life  and a 4.2-fold increase in tumour accumulation compared to the free enzyme. When PK1 (10 mg kg−1dox-equiv.) was injected i.v. into C57 mice bearing subcutaneously (s.c.) palpable B16F10 tumours followed after 5 h by HPMA copolymer-cathepsin B there was a rapid increase in the rate of dox release within the tumour . When PK1 and the PDEPT combination were used to treat established B16F10 melanoma tumour , the antitumour activity (T/C%) seen for the combination PDEPT was 168% compared to 152% seen for PK1 alone, and 144% for free dox. Also, the PDEPT combination showed activity against a COR-L23 xenograft whereas PK1 did not. PDEPT has certain advantages compared to ADEPT and GDEPT. The relatively short plasma residence time of the polymeric prodrug allows subsequent administration of polymer-enzyme without fear of prodrug activation in the circulation and polymer-enzyme conjugates have reduced immunogenicity. This study proves the concept of PDEPT and further optimisation is warranted. © 2001 Cancer Research Campaign   http://www.bjcancer.comPolymer-directed enzyme prodrug therapy (PDEPT) is a novel two-step antitumour approach using a combination of a polymeric prodrug and polymer-enzyme conjugate to generate cytotoxic drug selectively at the tumour site. In this study the polymeric prodrug N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-Gly-Phe-Leu-Gly-doxorubicin conjugate PK1  was selected as the model prodrug, and HPMA copolymer-cathepsin B as a model for the activating enzyme conjugate. Following polymer conjugation (yield of 30–35%) HPMA copolymer-cathepsin B retained ~20–25% enzymatic activity in vitro. To investigate pharmacokinetics in vivo,


Extracts from sea urchin intestine were screened for new anti-tumour drugs. Four glycolipids, 3'-sulphonoquinovosyl-1', 2'-diacylglyceride (A-4), 3'-sulphonoquinovosyl-1'-monoacylglyceride , NeuGc2-6Glc(beta)1-1ceramide (A-6) and HSO3-8NeuGc2-6Glc(beta)1-1ceramide (A-7), were isolated from the intestine of sea urchin, Strongylocentrotus intermedius, and characterized by means of proton nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. When tested for cytotoxic activity against tumour cells in vitro, A-5 showed significant activity, but A-4, -6 and -7 did not. In addition, the hydrophilic derivatives of A-4 or -5 had no cytotoxicity. Furthermore, the anti-tumour effects on nude mice bearing solid tumours of a human lung adenocarcinoma cell line A-549 were evaluated in vivo using A-4 and -5. As a result, A-5 was found to be significantly effective in suppressing the growth of solid tumours, whereas A-4 had no effect. Pathologically, the solid tumours showed haemorrhagic necrosis areas after treatment with A-5. In this study, we have demonstrated the anti-tumour effect of sulphonoquinovosyl-lysoglyceride (A-5), which provides important information that this sulpholipid could be a useful drug for cancer chemotherapy.


Vascular response was monitored using intravital microscopy and tumour cure was monitored by following regrowth over 42 days. For treatment at 5 or 30 min after BPD injection, blood flow stasis was limited to tumour microvasculature with lesser response in the surrounding normal microvasculature, indicating selective targeting for damage. No acute changes were observed in vessels when light was given 180 min after BPD injection. Tumour regression after light treatment occurred in all animals given PDT with BPD. Long-term tumour regression was greater in animals treated 5 min after BPD injection and least in animals given treatment 180 min after drug injection. The correlation between the timing for vascular damage and cure implies that blood flow stasis plays a significant role in PDT-induced tumour destruction. © 1999 Cancer Research CampaignBenzoporphyrin derivative monoacid ring A  is currently under investigation as a photosensitizer for photodynamic therapy (PDT). Since BPD exhibits rapid pharmacokinetics in plasma and tissues, we assessed damage to tumour and muscle microvasculature when light treatment for PDT was given at short times after injection of photosensitizer. Groups of rats with chondrosarcoma were given 2 mg kg


Cellular populations in the squamous cell carcinoma SCCVII, growing in C3H/HeN mice given Photofrin, were examined at various time intervals during the photodynamic light treatment and up to 8 h later. Cell populations present within excised tumours were identified by monoclonal antibodies directed against cell type-specific membrane markers using a combination of the indirect immunoperoxidase and Wright staining or by flow cytometry. Photofrin-based photodynamic therapy (PDT) induced dramatic changes in the level of different cellular populations contained in the treated tumour. The most pronounced was a rapid increase in the content of neutrophils, which increased 200-fold within 5 min after the initiation of light treatment. This was followed immediately by an increase in the levels of mast cells, while another type of myeloid cells, most likely monocytes, invaded the tumour between 0 and 2 h after PDT. The examination of cytolysis of in vitro cultured SCCVII tumour cells mediated by macrophages harvested from the SCCVII tumour revealed a pronounced increase in the tumoricidal activity of tumour-associated macrophages isolated at 2 h post PDT. It seems, therefore, that the PDT-induced acute inflammatory infiltration of myeloid cells into the treated tumour is associated with functional activation of immune cells.


The effect of nitric oxide-dependent vasodilators on vascular resistance of tumours and normal tissue was determined with the aim of modifying tumour blood flow for therapeutic benefit. Isolated preparations of the rat P22 tumour and normal rat hindlimb were perfused ex vivo. The effects on tissue vascular resistance of administration of sodium nitroprusside (SNP) and the diazeniumdiolate (or NONO-ate) NOC-7, vasodilators which act via direct release of nitric oxide (NO), were compared with the effects of acetylcholine (ACh), a vasodilator which acts primarily via receptor stimulation of endothelial cells to release NO in the form of endothelium-derived relaxing factor (EDRF). SNP and NOC-7 effectively dilated tumour blood vessels after preconstriction with phenylephrine (PE) or potassium chloride (KCl) as indicated by a decrease in vascular resistance. SNP also effectively dilated normal rat hindlimb vessels after PE/KCl constriction. Vasodilatation in the tumour preparations was accompanied by a significant rise in nitrite levels measured in the tumour effluent. ACh induced a significant vasodilation in the normal hindlimb but an anomalous vasoconstriction in the tumour. This result suggests that tumours, unlike normal tissues are incapable of releasing NO (EDRF) in response to ACh. Capacity for EDRF production may represent a difference between tumour and normal tissue blood vessels, which could be exploited for selective pharmacological manipulation of tumour blood flow.


Radioactively labelled (114mIndium) single chain antibody fragments-liposomes accumulated in the tumours at 2–3-fold higher concentrations during the first 2 h after i.v. injection compared to unmodified liposomes. After 6–24 h both liposome types were found in similar amounts (8–10% injected dose g−1) in the tumours. Animals treated i.v. with single chain antibody fragments-liposomes containing the new cytotoxic agent 2′-deoxy-5-fluorouridylyl-N4-octadecyl-1-β-D-arabinofuranosylcytosine  showed a reduction of tumour growth by 62–90% determined on days 5 and 8, respectively, compared to animals receiving control liposomes. Histological analysis revealed a marked reduction of F9 tumour cells and excessive deposition of fibronectin in the extracellular matrix after treatment with single chain antibody fragments-2-dioxy-5-fluorouridylyl-N4-octadecyl-1-β-D-arabinofuranosylcytosine-liposomes. Single chain antibody fragments-liposomes targeted to ED-B fibronectin positive tumours therefore represent a promising and versatile novel drug delivery system for the treatment of tumours.We prepared small unilamellar liposomes derivatised with single chain antibody fragments specific for the ED-B domain of B-fibronectin. This extracellular matrix associated protein is expressed around newly forming blood vessels in the vicinity of many types of tumours. The single chain antibody fragments were functionalised by introduction of C-terminal cysteines and linked to liposomes via maleimide groups located at the terminal ends of poly(ethylene glycol) modified phospholipids. The properties of these anti-ED-B single chain antibody fragments-liposomes were analysed British Journal of Cancer (2002) 87, 106–112. doi:10.1038/sj.bjc.6600423www.bjcancer.comCancer Research UK© 2002  Specific targeting of cytotoxic compounds to the tumour vasculature and the surrounding stroma of growing tumours would offer various advantages compared to conventional systemic chemotherapy. Fibronectin (Fn), in particular the B-Fn isoform with the ED-B oncofetal domain represents such a target protein that is expressed in and around neoplastic blood vessels during tumour growth and angiogenesis . ED-B FnCarnemolla characterised a single chain Fv antibody fragment (scFv) binding to the human ED-B domain of the B-Fn isoform using phage display technology cyclohexane-1-carboxylate (sulfo-SMCC) for the introduction of maleimide, allowing stable covalent S-C-linkage with C-terminal cysteine modified anti-ED-B scFv , a new amphiphilic compound with high cytotoxic activity (Specific binding of the anti-ED-B-scFv-liposomes (scFv-liposomes) was demonstrated on the ED-B expressing colorectal carcinoma cell line Caco-2 (2) was from Shearwater Polymers, Inc. . 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) and maleimide-BODIPY  were from Molecular Probes . 5-FdU-NOAC was synthesised as described by 114mInCl3 was from NEN Life Science Products  and [5-3H]-N4-octadecyl-1-β-D-arabinofuranosylcytosine (5-3[H]-NOAC) was custom labelled by Amersham Int. . Sulfo-SMCC was from Pierce . Dulbecco's modified Eagles medium (DMEM), foetal bovine serum and all medium supplements were from Gibco BRL . All buffer salts and other chemicals were of analytical grade and obtained from Fluka or Sigma .Soy phosphatidylcholine (SPC) was obtained from L Meyer . Cholesterol (CHOL) was purchased from Fluka . Methoxy-poly(ethylene glycol)-phosphatidylethanolamine (PE-PEG-OMet) was from Sygena . Amino-poly(ethylene glycol)-phosphatidylethanolamine  and the murine F9 teratocarcinoma cell line  was maintained in DMEM plus 10% heat-inactivated foetal bovine serum, 1% L-glutamine, 1% nonessential amino acids, 1 m2 (scFv modified liposomes) at a molar ratio of 1 : 0.2 : 0.07. For the in vitro binding experiments liposomes were labelled with the lipophilic dye DiO (0.004 mol parts referred to SPC). Maleimide-BODIPY was used for the determination of the modification efficiency of NH2-PEG-PE. For therapeutic studies 5-FdU-NOAC (0.075 mol parts referred to SPC) and 5-3[H]-NOAC as trace label for the drug were added to the lipid mixture.The liposomes were composed of SPC : CHOL : PE-PEG-OMet (unmodified liposomes) or PE-PEG-NHM, pH 7.4) through Nuclepore™ membranes  of 0.2 and 0.1 μm pore diameter with a Lipex™ extruder . Size and stability of the liposomes were analysed with a particle sizer . For the biodistribution experiments liposomes containing A23187 ionophore (0.001 mol parts referred to SPC) and nitrilotriacetic acid (1 mM) were labelled with 114mInCl3 as described by 114mInCl3  was incubated with unmodified control liposomes for 30 min at 60°C and with scFv-liposomes for 2 h at RT. Free 114mIn3+ was removed by size exclusion chromatography on a Sephadex G-50 column . 114mIn entrapment was determined with a gamma counter .All small unilamellar liposomes (SUV) were prepared by sequential filter extrusion of multilamellar liposomal preparations in phosphate buffer  in HBSE buffer . After reduction of the scFv dimer (0.5 mg ml−1) in HBSE with a 2 mM final concentration of tributylphosphine (TBP) for 4 h at 4°C sulfo-SMCC modified liposomes in HBSE were added and incubated for 20 h at 4°C under an argon atmosphere. Modified liposomes and non-reacted scFv were separated on a metrizamide gradient by ultracentrifugation  and incubated for 30 min at 37°C. Caco-2 and Co-115 cells (3×105 cells well−1) were plated and cultured for 48 h in a humidified 5% CO2 atmosphere at 37°C. Washed cells were incubated for 30 min at 4°C with 100 μl DiO labelled liposomes. Washed cover slips were removed, treated with 10% glycerine in PB, placed on glass slides and analysed on a fluorescence microscope (Leica DLMB). As negative controls the cells were incubated with unmodified fluorescence labelled liposomes.Cover slips were placed into 12 well plates, coated with 100 μl well7 F9 cells per 50 μl PB s.c. on both sides of the back. As tumours had reached sizes of 0.5–1.0 cm in diameter, 200 μl 114mIn labelled liposomes containing 5 mg SPC and 37.5 μg scFv in PB were injected i.v. Five minutes, 1, 2, 6 and 24 h later the animals were anaesthetised, and sacrificed by heart puncture and blood, heart, lung, liver, spleen, kidneys and tumours were removed, weighed and the radioactivity measured by gamma counting . Blood correction factors were applied to all organ samples  were injected s.c. on the abdominal side of CD-1<R>-nu/nu mice. Mice (5–10 per group) were treated i.v. as the tumours had reached diameters of 6–8 mm with scFv-5-FdU-NOAC-liposomes, 5-FdU-NOAC-liposomes, scFv-liposomes, unmodified liposomes or scFv-dimers every 24 h for 5 days. Each dose (0.2 ml) contained 0.6 mg 5-FdU-NOAC (30 mg kg−1 per dose) or corresponding amounts of lipids or protein. Tumour growth was measured in a blinded fashion with a caliper every day and volumes calculated using the following equation: V=π ab2/6 . Tumour v w−1 values were converted into percent change from baseline using equation Vt×100/V0 . Mice were sacrificed 8 days after onset of treatment and tumours were dissected and weighed.F9 cells (10Samples of F9 tumours were collected 72 h after one treatment (0.6 mg 5-FdU-NOAC) and histology was done as described by 2 were calculated. The efficiency of the modification with sulfo-SMCC was determined by fluorescence labelling of liposomes with maleimide-BODIPY resulting in 60% of maleimide modified PEG-NH2-groups (data not shown).Total liposome numbers and the number of all lipophilic molecules per liposome were calculated from the mean diameter obtained from laser light scattering data and from liposome vesicle geometry parameters as described by M tributylphosphine for 4 h at 4°C produced active and stable monomers. Consequently, the scFv were coupled to the distal end of the PEG chain via the cysteine thiol groups in the presence of the reducing agent. Non-reacted scFv were separated from liposomes on a metrizamide gradient. Fractions collected from the gradient were analysed by gel electrophoresis, followed by Western blot analysis (data not shown). Numbers of protein molecules linked per liposome were calculated by determining the amount of lipid based on DiO fluorescence and the protein concentration. To liposomes of 100±23 nm diameter an average of 220 scFv were attached, corresponding to a coupling efficiency of 15±3%.In the absence of reducing agents the anti-ED-B scFv constructs form dimers due to disulfide bridge formation  followed by doxorubicin (IC50=10 μM) and 5-FdU-NOAC (IC50=10 μM). We chose 5-FdU-NOAC because due to its lipophilic properties this new duplex drug can be added directly to the lipids during liposome preparation  on days 5 to 8. However, the statistical comparison between 5-FdU-NOAC liposomes and scFv-5-FdU-NOAC liposomes revealed no significant difference (P=0.14). After 8 days the tumours of untreated control mice had reached a large size requiring the sacrifice of the animals. The tumours were immediately excised and weighed. Calculated by weight a 90% reduction of tumour mass was found with the scFv-5FdU-NOAC-liposomes.Different cytotoxic agents  were tested on F9 cells using a cell viability test (data not shown). The most active compound was mitoxantrone . In contrast, anti-ED-B-Fn staining showed distinct differences between unloaded and 5-FdU-NOAC-liposomes  in the tumours. This may be explained by two observations, firstly the well described high uptake of pegylated liposomes into solid tumours that is mainly a consequence of the long blood circulation times (−1 after 1 h (in vivo. The rapid clearance of scFv-liposomes from circulation is due to the opsonisation of protein coated liposomes followed by the MPS uptake (With such fluorescently labelled scFv-liposomes we showed strong binding -2 cells . The bio-2 cells  and 3B. on times . Secondlfter 1 h . This reIn the therapy studies the mice treated with specific scFv-5-FdU-NOAC-liposomes showed a remarkable reduction in tumour size. This enhanced cytotoxic effect of scFv-5-FdU-NOAC-liposomes could be explained by the specific binding to ED-B Fn causing longer lasting liposome retention in the tumour tissue and promoting deeper penetration into the extracellular matrix. Nevertheless, together with a reduction of the number of scFv molecules per liposome, optimisation of the therapy schedule and the use of other drugs might lead to improved therapeutic modalities.The histological examination of the F9 tumours treated with 5-FdU-NOAC containing liposomes revealed that the cytotoxic effect mainly affected tumour cells. Examination of the Fn distribution in the tumours by scFv L19 staining exposed a remarkable deposition of Fn after treatment with the scFv-5-FdU-NOAC liposomes  that wasThe cytotoxic activity of non-targeted 5-FdU-NOAC-liposomes is noteworthy. 5-FdU-NOAC is a drug representative for a series of new amphiphilic/lipophilic nucleoside duplex drugs that were developed by us (In conclusion, our experiments are the first to our knowledge to show inhibition of tumour growth by targeting immunoliposomes to a protein that is not exclusively expressed on tumour cells but also in the stroma and on endothelial cells of the tumour vasculature. Keeping in mind that the ED-B domain of fibronectin is conserved between mouse and humans, ED-B Fn specific scFv-liposomes containing cytotoxic and/or anti-angiogenic compounds represent a promising approach for the therapeutic targeting of solid tumours in humans.


The extent of the angiogenic response induced by T47D spheroids implanted into the dorsal skinfold chamber in nude mice was measured in vivo and compared to that induced by spheroids infiltrated with human macrophages prior to implantation. Our results indicate that the presence of macrophages in spheroids resulted in at least a three-fold upregulation in the release of vascular endothelial growth factor (VEGF) in vitro when compared with spheroids composed only of tumour cells. The angiogenic response measured around the spheroids, 3 days after in vivo implantation, was significantly greater in the spheroids infiltrated with macrophages. The number of vessels increased , were shorter in length  with an increased number of junctions  all parameters indicative of new vessel formation. This is the first study to demonstrate a role for macrophages in the initiation of tumour angiogenesis in vivo.An  Angiogenesis, the development of new blood vessels from an existing vascular network, is a prerequisite for tumour growth beyond 2 mm in diameter, as it ensures the adequate supply of oxygen and nutrients for tumour cell expansion and spread . Hypoxiavitro studies have shown that TAMs secrete growth factors that are mitogenic for both tumour cells and endothelial cells, in addition to stimulating the activity of a number of proteolytic enzymes in breast cancer (in vitro (Tumour associated macrophages (TAMs) are widespread in human breast carcinomas  cells. Human breast tumour spheroids were infiltrated with human monocytes in vitro, coated with alginate and implanted into dorsal skinfold chambers on nude mice. The resultant angiogenesis surrounding the spheroids was quantified using image analysis, and compared with spheroids consisting of tumour cells alone.Despite the increasing body of evidence, both experimental and clinical, implicating macrophages in breast tumour angiogenesis, there have been no reports of 2 added to make platelet rich plasma-derived serum (PRPDS). The blood cells were layered onto a Ficoll gradient (Amersham Biosciences) and after centrifugation the mononuclear cell layer collected. The cells were further purified using CD14 magnetic beads according to the manufacturers instructions (Miltenyi Biotec). Briefly, the cells were incubated with the labelled beads at 4°C for 20–30 min, washed in ice-cold PBS+0.5% BSA, 2 mM EDTA, and separated from unlabelled cells using a magnetic column.Peripheral blood mononuclear cells were isolated from normal healthy volunteers with Local Ethics Committee approval, LREC 02/303. Blood was collected into sodium citrate  tubes and after centrifugation serum was collected and CaClμl of 1.5% high-gel point agarose per well. The human T47D breast tumour cell line was subcultured and 104 cells plated per well in 200 μl culture medium consisting of RPMI (Sigma), 10% foetal calf serum FCS, (Sigma), 100 U ml−1 penicillin (Gibco), 100 μg ml−1 streptomycin (Gibco) and 2 mM glutamine (Gibco). The plates were incubated at 37°C, 5% CO2 for 4 days, thereafter the culture medium was changed on alternate days by replacing 100 μl of medium with 100 μl fresh culture medium. Preliminary studies have established that between 20–24 days in culture the spheroids are approximately 600–800 μm in diameter and have a necrotic core (unpublished results).96-well tissue culture plates (Corning) were coated with 100 in vivo implantation using the following protocol. Spheroids were cultured for 14–18 days and human monocytes (105 spheroid−1 well−1) were added and allowed to infiltrate the spheroids for 24 h. In order to prevent autologous (i.e. murine) macrophages from infiltrating the spheroid following implantation in vivo, the spheroids were coated with alginate  were added to the culture in RPMI with 5% PRPDS, penicillin (100 U ml−1), streptomycin (100 mg ml−1) and glutamine (2 mM). The spheroids were collected on days 1, 4, and 7 after monocyte addition, formalin-fixed and embedded in paraffin. Sections were stained with anti-human CD68  a specific monocyte/macrophage marker, and the number of monocytes/macrophages in each spheroid section counted. For each time point at least five different spheroids and three sections from each spheroid were used to quantify the number of macrophages present.T47D spheroids were established in culture and allowed to grow for 17 days before purified monocytes  to spheroid cultures, before and after alginate coating (days 15–19) and immediately before spheroid implantation into the skinfold chamber model (days 21–24). At all times, control spheroids (no monocyte infiltration) were cultured in parallel and medium was collected simultaneously. Immediately after collection, the medium was centrifuged to remove cell debris and stored in aliquots at −20°C until assayed. The ELISAs were performed using commercially available kits (R&D Systems) and carried out according to the manufacturers protocol.μm sections. The sections were dewaxed in xylene and absolute alcohol before endogenous peroxide was blocked with hydrogen peroxide. Antigen retrieval was achieved by incubating in protease type XXIV (Sigma) at 37°C for 20 min. After incubation in normal serum, anti-CD68 (Dako) was diluted 1 : 100 in PBS added to sections and incubated overnight at 4°C. A Vectastain kit (Vector Laboratories) was used for secondary antibody application and detection. Spheroids that had been implanted in the dorsal skinfold chamber were collected at the end of each experiment and fixed immediately in zinc chloride saline  and with anti-platelet cell adhesion molecule antibody .Spheroids (with and without monocytes) were collected directly into buffered formalin, processed, paraffin–embedded, and cut into 5 e saline , processn=10) were obtained from the University of Sheffield Field Laboratories and were allowed food and water ad libitum. All procedures were performed in accordance with UK legislation under Home Office Project Licence PPL 40/2343.Adult, male MF-1 nude mice . The mice were placed on a heated pad, and a double layer of skin extended perpendicular to the dorsum. A circular area of skin and tissue was removed from one side to expose a single layer of striated muscle, which was then sandwiched between two symmetrical titanium frames. The tumour spheroid (approximately 600 μm) was placed in close proximity to a blood vessel immediately before sealing the chamber with a glass cover slip , displayed on a monitor  and recorded on video  tape for off-line computerised analysis . The day of surgery was designated day 0 and recordings were made on days 3 and 7.The dorsal skinfold chamber manufactured from titanium  was implanted into the animal under hypnorm/diazepam anaesthesia , an increase indicating endothelial proliferation and differentiation, the total number of vessels per unit area (vessel density) with and without blood flow indicating the new structures are functional and the number of junctions (branch points) indicates vascular network complexity. All images were collected for analysis using a 10 × objective and the same area was measured at each time point and for each animal. A formal assessment of blood flow was not made. As the spheroids implanted in this series of experiments were coated with alginate it was not possible to measure the growth of new blood vessels into each spheroid.The image analysis programme Angiosys  was used to assess new vessel formation. The area immediately surrounding the spheroid and at least three randomly selected sites, distant to the spheroid, were analysed. The two groups  were analysed at 3 and 7 days after implantation of the spheroid; the parameters measured were mean vessel length . Thus macrophages within the alginate-coated spheroid were releasing VEGF and therefore the contribution to the initiation of tumour angiogenesis could be estimated in the in vivo model. A homogeneous distribution of macrophages surrounding the hypoxic centre was observed in the majority of spheroid sections assessed. Three sections from five different spheroids were quantified and used to estimate the number of infiltrated macrophages per spheroid, which were approximately 400 macrophages  as was the number of junctions  implying that new vessels had branched from existing vessels. The mean tubule length measured at these times was less on day 7 than on day 3  indicating that new shorter blood vessels had sprouted due to the stimulatory effects of the tumour spheroid. However, by day 7 the differences between spheroids with and without macrophages were no longer significant. (The data collected from off-line analysis indicates that angiogenesis were stimulated by the breast tumour spheroids in the presence or absence of macrophages  but the ificant.  A formalin vivo study by This is the first study to demonstrate that macrophages may modulate tumour angiogenesis in the early stages of development, with increased number of vessels and branches. A number of studies have shown that macrophages congregate in large numbers in the hypoxic areas of many solid tumours, which may be due to the influence of hypoxia on the chemoattractant signalling cascade in the tumour microenvironment (. Macrophα staining evident surrounding the necrosis. This was similar both in the presence and absence of macrophages and therefore is unlikely to be due to a direct anti-tumour action of the macrophages. VEGF is a potent endothelial cell mitogen and has previously been shown to be released from both T47D tumour cells and macrophages (in vivo implantation, thereby isolating them from the host immune cells. Previous studies demonstrated the coating has no effect on cellular growth or proliferation, and indicated that in the first week after implantation in vivo, mononuclear cells migrated to the rim of the alginate spheroid but there was no penetration, and over the following nine weeks, the number of migrating cells gradually decreased (2 in medullary carcinomas and 343 mm−2 in ductal carcinomas.In the current study human monocytes isolated from peripheral blood were cocultured with human breast T47D tumour spheroids allowing monocyte migration into the tumour spheroid where differentiation into tumour-associated macrophages occurs (ecreased . The perin vivo. The hypothesis of the current study is that the presence of macrophages increase the angiogenic potential of tumour spheroids through the increased production of VEGF. Thus to evaluate any role for macrophages in the initiation of tumour angiogenesis, an in vivo model involving the implantation of breast tumour spheroids into a dorsal skinfold chamber on nude mice has been developed in our laboratory. The mice are immunodeficient, with an absence of T cells but a normal complement of B cells and elevated levels of both natural killer cells and macrophages. Macrophages in nude mice appear more potent than those from mice with a normal thymus (The role of macrophages has not been extensively studied l thymus .in vivo models (Following implantation, parameters measured to determine any effect of the macrophages on angiogenesis included vessel length, vessel numbers, junctions or branch points, in areas directly adjacent to, in addition to distant from the spheroid. Mean vessel diameter is not presented as often distant sites included large host vessels present throughout the procedure, which skewed analysis. The angiogenesis parameter measurements three days after implantation of the tumour spheroid indicated that macrophages play a role in the initial angiogenic response, with significant increases in the density of blood vessels and the number of junctions, with a reduction in vessel length in comparison to control spheroids composed solely of tumour cells. The shorter vessel length was an unexpected and as yet unexplained finding although vessels were generally of a length found in other o models .In conclusion therefore, the current study demonstrates that macrophages present within a solid tumour may significantly contribute to the initiation of angiogenesis. However, in the absence of macrophages, tumour cells produce the necessary stimuli to initiate tumour angiogenesis but initiation is delayed. Further long-term studies, using a syngenic murine model and uncoated spheroids infiltrated with host murine macrophages, allowing the tumour stroma to play a role, or with host macrophage function inhibited, would allow the contribution of macrophages to the maintenance of angiogenesis, endothelial cell survival and tumour growth to be determined. In addition the model could be used to assess the future of the macrophage as a target for tumour therapy.