The ideal cancer treatment should both destroy the primary tumour and at the same time educate the immune system to recognise the tumour as foreign so that distant metastases will also be eradicated. Photodynamic therapy (PDT) involves the i.v. administration of photosensitisers followed by illumination of the tumour with red light producing reactive oxygen species that eventually cause vascular shutdown and tumour cell death by apoptosis and necrosis. Anti-tumour immunity is stimulated after PDT due to the acute inflammatory response, generation of tumour-specific antigens, and induction of heat-shock proteins. Green fluorescent protein (GFP) is used as an optical reporter to noninvasively image the progression of mouse tumours, and in addition, may act as a foreign (jellyfish) antigen. We asked whether GFP-expressing tumours could be used to monitor the response of tumour-bearing mice to PDT, and whether the tumour response differed when a nonimmunogenic tumour cell line was transduced with GFP. We injected RIF-1 or RIF-1 EGFP  cells in the leg of C3H/HeN mice and both the cells and tumour grew equally well. We used PDT with benzoporphyrin derivative and a short drug-light interval. There were complete cures and 100% mouse survival of RIF-1 EGFP while RIF-1 wild-type tumours all recurred. Cured mice were resistant to rechallenge with RIF-1 EGFP cells and a rechallenge with wild-type RIF-1 cells grew significantly slower. There was also slower RIF-1 EGFP rechallenge growth but no rejection when RIF-1 EGFP tumours were surgically removed. There was a low rate of PDT cure of tumours when RIF-1 cells were transduced with an empty retroviral vector. The presence of antibodies against EGFP in mouse serum suggests EGFP can act as a foreign antigen and PDT can then stimulate a long-term memory immune response. It was estimated that approximately 563 700 Americans would die of cancer in 2004 corresponding to over 1500 deaths per day (Photodynamic therapy (PDT) uses a nontoxic photoactivatable dye or photosensitiser (PS) in combination with harmless visible light that produces reactive oxygen species and destroys tumour tissue . MechaniAequorea victoria  was originally isolated from the jellyfish, victoria . Enhancevictoria , 2002a h−1 penicillin and 100 μg ml−1 streptomycin. They were collected for injection by washing with PBS without Ca2+ and Mg2+, and adding trypsin-EDTA to the plate for 10 min at 37°C.The radiation-induced fibrosarcoma (RIF-1) tumour cells originally characterised by Escherichia coli DH5a . The plasmid was then purified with the Plasmid Kit from Qiagen  as instructed. A packaging cell line  was transfected with the plasmid vector using Lipofectamine 2000  as described for the reagent. Medium containing the retrovirus was collected after 48 h post-transfection, filtered through a 0.45 μm membrane, and used to infect the RIF-1 cells, as described in the retroviral gene expression user manual. Radiation-induced fibrosarcoma cells permanently expressing EGFP (RIF-1 EGFP) were initially cultured in 2 mg ml−1 G418 and selected for EGFP expression by limiting dilution and fluorescence microscopy. This was carried out in order to pick a stable and bright level of fluorescence. Cells were not routinely maintained with G418 but were used for a maximum of six passages and EGFP expression levels were checked at passage 6 by FACS analysis. Additional RIF-1 cells were transduced with the empty retroviral vector  lacking the gene for EGFP and selected and routinely cultured in the presence of 2 mg ml−1 G418. These were termed RIF-1 EPV.The retroviral plasmid vector  expressing the enhanced GFP (EGFP) was purchased from BD Clontech , and propagated in −1 ketamine, 10 mg kg−1 xylazine). One million RIF-1 cells or RIF-1 EGFP cells were injected subcutaneously in one mid-thigh area suspended in 100 μl PBS. Tumours grew predictably in all mice and reached a size of 5–6-mm diameter 8–9 days after injection at which time they were used for PDT. Some mice had their tumours removed by surgical amputation of the tumour bearing leg at the same time point (5–6 mm diameter).All animal experiments were approved by the Subcommittee on Research Animal Care of Massachusetts General Hospital and were in compliance with NIH guidelines. Male C3H/HeN mice , weighing 20–25 g were shaved on the right thigh and depilated with Nair . Mice were anaesthetised with an i.p. injection of ketamine/xylazine cocktail , incubated at room temperature for 90 min. The wells were then blocked by incubating with PBS containing 1% BSA for 1 h at room temperature, and washed three times with PBS containing 0.05% Tween 20 (PBS-T). Mouse serum (10 μl) was diluted into 100 μl PBS-T and added to the wells, incubated for 2 h at room temperature. Then the serum was removed and the wells washed three times with the PBS-T. Captured mouse anti-EGFP was detected with an alkaline phosphatase-conjugated secondary antibody  and D-nitrophenylphosphate (Sigma). Optical absorbance at 405 nm was read at 60 min on a plate reader (Molecular Dynamics). A calibration curve was constructed using known amounts of mouse IgG coated on wells of a plate and the same alkaline phosphatase-conjugated secondary antibody, that allowed the OD at 405 nm to be converted to μg ml−1 IgG.An ELISA assay for mouse anti-EGFP detection was modified from that of −1 ketamine, 5 mg kg−1 xylazine) and were imaged in a macrofluorescence imaging system . Incandescent white light  was filtered through a 450–490 band pass filter and a liquid light guide to provide illumination from two parallel angled light diffusers positioned 25 cm above the mouse. Emission light passed through a 500-nm long pass filter into an Optronics CE  colour charge-coupled device camera via a f1.4 zoom lens. The camera was controlled by Magnafire SP software (Optronics) and an acquisition time of 2.468 s and a white balance of 2800 K were used to capture images of 1280 by 1024 pixels. Adobe Photoshop  was used to compile time course composite images.Mice bearing GFP expressing tumours were lightly anaesthetised with an i.p. injection of ketamine/xylazine cocktail  emitting light at 690 nm (±2 nm). The laser was coupled into a 400-μm fibre via a SMA connector and light from the distal end of the fibre was focused into a uniform spot with an objective lens . The spot had a diameter of 1.2 cm and was positioned so that the entire tumour and a surrounding 2–3 mm of normal tissue was exposed to light. Mice were anaesthetised as described above and the tumour bearing leg positioned under the spot. A total fluence of 150 J cm−2 was delivered at a fluence rate of 100 mW cm−2. Some mice had their RIF-1 or RIF-1 EGFP tumours illuminated without having received a prior injection of BPD. At the completion of the illumination mice were allowed to recover in an animal warmer until they resumed their normal activity.BPD  was a generous gift from QLT Inc.  as a lyophilised powder, which was reconstituted with 5% dextrose solution and injected at a dose of 2 mg kga and b) were measured with vernier calipers. The tumour volume was calculated according to the formula, volume=4/3 π{(a+b)/4}3. Tumours were treated when 5–6 mm in diameter (volume ≈70–110 mm3). Mice were imaged in the GFP camera if their tumour was EGFP expressing. Mice were killed according to the protocol when the tumours reached a largest diameter of 1 cm. Mice were considered cured when the tumour did not return after 60 days. Cured mice or surgically amputated mice were rechallenged with one million tumour cells (RIF-1 EGFP or RIF-1) injected in the opposite leg to the one that had previously borne the primary tumour.Mice were examined three times a week. They were weighed and the orthogonal tumour dimensions . The tumour growth curves were analysed by transforming the data to a logarithmic scale and comparing the slopes. P-values of <0.05 were considered significant.All values are expressed as±s.e. of the mean. Comparison between two means was carried out using the Mann–Whitney It was necessary to ensure that the stable expression of the EGFP gene via a retrovirus, did not have any deleterious effects on either the growth rate of the tumour cells in tissue culture, or the growth rate of the tumours when subcutaneously implanted into C3H mice. The doubling times of the RIF-1, RIF-1 EGFP and RIF-1 EPV cells in tissue culture were not statistically different (data not shown). As can be seen from Fluorescence imaging was used to noninvasively visualise the progression of the EGFP-expressing tumours. In μl) was withdrawn via the orbital plexus from mice bearing RIF-1 or RIF-1 EGFP tumours, allowed to clot and serum obtained. Serum samples were analysed for the presence of anti-EGFP antibodies as described in Materials and Methods. Blood (100 −1 i.v. followed after 15 min by 150 J cm−2 delivered at 100 mW cm−2). At 1 to 2 days after PDT the mice exhibited a black eschar confined to the area formerly occupied by the tumour, It was difficult to determine how much tumour was left under the black eschar but in all mice the volume of the tissue measured decreased after PDT. However, in all mice the tumours regrew, frequently forming a ring of viable rapidly growing tumour outside the black eschar. When RIF-1 EGFP tumours were treated under the same conditions with BPD-PDT we observed interesting and surprising differences in the responses. Firstly the tumour shrinkage was more marked with RIF-1 EGFP tumours than was found with RIF-1 tumours. Secondly, the subsequent behavior of the tumours was very different. The RIF-1 EGFP tumours disappeared completely by day 20 and did not return during the 40 days they were followed. The progress of one mouse bearing a RIF-1 EGFP tumour treated with BPD-PDT and monitored with fluorescence imaging is depicted in Wild-type RIF-1 tumours were treated with PDT at days 8–9 (when tumours had a diameter of 5–6 mm). We used BPD . There was no difference between eviously . When miP<0.001 by comparing slopes of logarithmic transformed data). Five mice that had been cured of RIF-1 EGFP tumours and rejected a subsequent rechallenge with RIF-1 EGFP tumour cells were rechallenged with one million wild-type RIF-1 cells together with naïve mice. The RIF-1 tumours grew in all the mice but again the rate of growth of RIF-1 cells in mice cured of RIF-1 EGFP tumours by PDT was significantly slower than the rate in naïve mice (P<0.001 by comparing slopes of logarithmic transformed data) than in naïve mice , while all of the surgically cured mice (five out of five), and all of the naïve mice that were challenged with RIF-1 EGFP cells grew tumours (five out of five). However, the growth rate of the surgically cured mice rechallenged with RIF-1 EGFP was significantly slower than the growth rate of naive RIF-1 EGFP tumours  is processed and presented through MHC I with generation of tumour-specific CTL. This system has been used to test several approaches involving cancer vaccination strategies  also can induce a strong cellular immune response against the antigen  when tumours formed from RIF-1 cells that had been transduced with the empty retroviral vector as a control (RIF-1 EPV, The generation of a long-term memory immune response as demonstrated by the resistance of cured mice to rechallenge with RIF-1 EGFP cells was not unexpected. Reports have demonstrated that the production of anti-tumour immunity by curing tumours in mice either by PDT (There is a considerable amount of work remaining to be done to understand these results. Our future work will be focused on the identification of the immune mechanisms behind the immunity generated by PDT against GFP and wild-type tumours. We need to do additional work to establish the role that EGFP is playing in comparison with the retroviral vector used to transduce the cells. However, the presence of anti-GFP antibodies before PDT suggests that the effect is primarily mediated by the foreign jellyfish antigen. We will investigate whether mice cured of RIF-1 EGFP tumours also have immunity against other EGFP-expressing tumours cells syngeneic to C3H mice.


Ten xenograft lines of human colonic and rectal carcinomas have been established in immune-suppressed mice. Mice bearing tumours in the 2nd to 6th passage were treated with maximum tolerated doses of 8 chemotherapeutic agents and tumour growth delay was estimated in terms of the number of volume doubling times gained by the treatment. The average response corresponded to a delay of only about 0.5 doubling times, but some tumour lines showed a good response to some agents. Twenty-three out of about 700 treated tumours failed to regrow. Statistical analysis showed no consistent difference in sensitivity among the tumour lines, but melphalan, 5-fluorouracil and hexamethylmelamine stood out as the most effective agents. A study of two-drug combinations showed that their order of administration had little effect on response. Only 4 of the patients who donated the xenografts were treated with chemotherapy, but among these there was some evidence that response in the xenografts correlated with response in the patient.


Early neoplastic lesions, primary tumours and metastases showed consistent and rapid PpIX accumulation compared to the normal surrounding tissues, as evidenced by red fluorescence (635 nm) when the tumours were directly illuminated with blue light (380–440 nm). Detection of mouse mammary tumours at the stage of ductal carcinomain situ by red fluorescence emissions suggests that enhanced PpIX synthesis is a good marker for early tumorigenic processes in the mammary gland. We propose the mouse models provide an ideal experimental system for further investigation of the early diagnostic and therapeutic potential of 5-ALA-stimulated PpIX accumulation in human breast cancer patients.A photodynamic technique for human breast cancer detection founded upon the ability of tumour cells to rapidly accumulate the fluorescent product protoporphyrin IX (PpIX) has been applied to transgenic mouse models of mammary tumorigenesis. A major goal of this investigation was to determine whether mouse mammary tumours are reliable models of human disease in terms of PpIX accumulation, for future mechanistic and therapeutic studies. The haeme substrate 5-aminolevulinic acid (5-ALA) (200 mg kg Synthesis of the fundamental co-factor haeme occurs as a multistep biochemical pathway within the cytoplasmic and mitochondrial compartments of all mammalian cells. Haeme synthesis shows controlled and complex regulation with prominent substrate inhibition at the first step of 5-aminolevulinic acid (5-ALA) production . ExogenoThe general property of PpIX loading by tumour cells, either inherently, or following 5-ALA dosing, has stimulated numerous basic research and clinical studies for potential therapeutic and diagnostic uses. PpIX is a natural photosensitiser that generates reactive oxygen intermediates following excitation with 635 nm light. Thus 5-ALA-stimulated PpIX loading has been evaluated for photodynamic therapy (PDT) to eradicate tumour cells  carries the human RAS oncogene  with mammary tissue-specific gene expression driven by the Whey acidic protein (Wap) promoter (Wap promoter (http://tumor.informatics.jax.org) via the Mouse Genome Informatics website (www.informatics.jax.org) of The Jackson Laboratory (Three transgenic mouse models of mammary tumorigenesis were used for this investigation: (1) Transgenic strain FVB.Cg-Tg(WapHRAS)69Lln Chr Yad libitum and HCl-acidified water (pH2.8–3.2) ad libitum to retard bacterial growth. Animals were weaned at 18–23 days of age and housed in same sex groups of 3–5 in 51 in2 (329 cm2) polycarbonate cages containing sterilised White Pine shavings. All animal procedures were approved by the Animal Care and Use Committee of The Jackson Laboratory.All mice were produced and housed in our research colony at The Jackson Laboratory under 14 h : 10 h light : dark cycles. Mice were provided with autoclaved NIH-31 diet   M) to achieve a final dose of 200 mg kg−1 of body weight. Animals were kept in their cages and maintained in a darkened room for a 60–75 min period prior to necropsy.5-ALA.HCl  was dissolved in sterile saline (0.85% NaCl) immediately prior to administration, and kept in a light-protected vial. All animals were administered a tail vein injection of 5-ALA solution  generated by a D-light system  to excite PpIX fluorescence. Digital images were recorded with a Leica DFX 350 camera mounted on a Wild M10 stereo-microscope  that was fitted with a 635 nm emission filter provided by Storz GmbH. Isolated lungs with mammary tumour metastasis were imaged with a 35 mm camera loaded with 160T slide film , that was fitted with the 635 nm emission filter. When fluorescent tumour foci were ⩽1 mm in size, blue background was digitally subtracted from the entire image to provide sufficient contrast for publication purposes .Following Isolated tumour foci were fixed overnight in Bouin's solution, and histological sections processed for hematoxylin and eosin (H&E) staining. For early neoplastic lesions of WapTag1 mammary glands, a minimum of 30 serial sections were examined to confirm that no mature carcinomas were evident.−1 collagenase type IA . Isolated cell suspensions for flow cytometry were generated by a 15-min-incubation in collagenase/PBS under foil cover and disaggregration via successive aspiration through 18 and 20 gauge needles. The cell suspensions were kept on ice, under foil wrap until fluorescence measurements with a FACSVantage SE/DiVa flow cytometer . PpIX excitation was achieved with a 415.4 nm Innova 90-2 Krypton laser. PpIX fluorescence emissions were recorded following reflection by a 610 nm short pass dichroic mirror to a 590 nm long pass filter . Acquistion and analysis of the data for 10 000 tumour cells were performed with FACSDiva ver. 4.0, FACS Convert ver. 1.0 and CellQuestPro ver. 4.0.1 .Mammary glands or palpable tumour foci from HRAS transgenic males and PyVT transgenic females and littermate controls were excised and minced with fine scissors in phosphate-buffered saline (PBS) containing 2 mg mlP<0.05 as the chosen level of statistical significance.The quantitative fluorescence flow cytometric data, representing a minimum of three independent replicate samples, was analysed by ANOVA with Statview software  with Mice bearing palpable mammary tumours were investigated for their PpIX accumulation response following 5-ALA administration. Mice were euthanised and the mammary tumours exteriorised to examine PpIX accumulation under blue light excitation. Mammary tumours in each of the transgenic models responded to 5-ALA administration with significant PpIX accumulation, as measured by tumour-selective red fluorescence relative to the normal surrounding tissues. Fluorescence was most pronounced in PyVT mammary tumours, but was clearly evident in tumours from each transgenic model, despite differences in histological subtype and the gender of the tumour-bearing animal . WapTag1ductal carcinomain situ (DCIS) with accompanying inflammation. Two examples of PpIX stimulated red fluorescence associated with DCIS are illustrated in Given the evidence for PpIX accumulation in palpable, mature mammary tumours, we administered 5-ALA to WaptTag1 transgenic female mice prior to the anticipated age for palpable tumour presentation, when early stages of tumour progression are predicted for this mouse model. In four independent WapTag1 females, 5-ALA stimulated PpIX accumulation permitted red fluorescence detection of areas histologically confirmed as vs control tumour-bearing males is shown in vs the normal surrounding tissues, we also compared the PpIX fluoresence emissions of normal mammary glands obtained from nontransgene carrier, virgin littermates of the PyVT females. Like the tumour cells, the normal mammary glands were either exposed to 5-ALA (200 mg kg−1) for 1 h in vivo or were obtained directly from unprimed control females. 5-ALA administration induced a significant, two-fold increase in red fluorescence emissions in the normal mammary gland, although this increase was significantly less than the seven-fold increase observed in 5-ALA-primed PyVT tumour cells, and did not interfere with the visual margins of differential red fluorescence observed between tumour cells and normal tissues in the intact animal  homogeneous mammary tumours of HRAS transgenic males and PyVT transgenic females. A comparison of the fluorescence emitted from isolated HRAS tumour cells derived from 5-ALA-primed t animal .To establish whether transgenic mouse models of mammary tumorigenesis are relevant models for human breast cancer in terms of their response to 5-ALA, we have evaluated three well-characterised transgenic strains that develop mammary tumours under powerful oncogenic stimulation – HRAS, PyVT and SV40 Tag – for their ability to accumulate PpIX following administration of 5-ALA. Our findings showed that the mammary tumours from each transgenic strain rapidly and selectively converted 5-ALA to PpIX, and showed five- to seven-fold enhanced fluorescence compared with surrounding normal tissues under blue light excitation. Similar to the primary mammary tumours, spontaneous metastatic lesions to the lungs of tumour bearing PyVT mice were readily demarcated by PpIX accumulation and red fluorescence under blue light, suggesting that the cellular mechanisms responsible for PpIX accumulation are retained within tumour populations that acquire metastatic competence. This follows the findings of PpIX accumulation in the primary tumours of each strain was consistent, despite fundamental differences in histological subtype, latency of onset, metastatic potential and gender of the tumour-bearing host. Thus, the transgenic models parallel the findings for human breast cancer cases in that the PpIX accumulation phenotype is not selective for any particular tumour histological subtype (A new avenue of clinical tumour imaging for human breast cancer has opened with the availability of fiberoptic ductoscopy (Confirmation that mouse models of both male and female mammary tumorigenesis parallel the clinical findings for human breast cancer in terms of PpIX synthesis could lead to new lines of investigation for the detection, diagnosis and therapy of breast cancer. One advantage of the mouse models of mammary tumorigenesis over cell culture models is that cellular events leading to the PpIX accumulation phenotype can be genetically and chronologically mapped according to the various stages of tumorigenesis. Furthermore, an investigation as to whether altered regulation of the haeme synthesis pathway is important for tumour development can be further addressed in genetically engineered mouse models of mammary tumorigenesis.


Extensive investigation of 3-(p-chlorophenyl)-2,3-dihydro-3-hydroxythiazolo-benzimidazole-2-acetic acid  in BDF1 mice implanted with Lewis lung tumour has shown that it is an effective anti-tumour and anti-metastatic agent. In vitro examination using HEp-2 human epidermal tumour cells has indicated that Wy-13,876 is not cytotoxic. When mice implanted with Lewis lung tumour and treated with Wy-13,876 are also injected with anti-thymocyte serum, an increase in lung metastases is observed suggesting that thymocyte activity is involved in the drug's mechanism of action. An increase in peripheral T lymphocytes observed in rats 18 h after a single oral dose of Wy-13,876 further supports this possibility. When Wy-13,876 is given to tumour -bearing mice in combination with low, ineffective doses of 5-fluorouracil or cyclophosphamide, further reduction of primary tumour growth is observed.


RNA extracted from the spleens of tumour-bearing (TLRNA) and tumour-immune (ILRNA) mice was shown to transfer to normal lymphocytes (NL) the ability to produce factors that blocked specific tumour-cell cytotoxicity and mediated specific antibody-dependent cell cytotoxicity (ADCC). Aliquots of normal C3H mouse lymphocytes were treated with TLRNA or ILRNA and cultured in vitro in the absence of tumour antigen. Supernatants were collected at 24h intervals and tested in a microcytotoxicity assay for blocking and ADCC activities. Factors that inhibited tumour destruction by specifically sensitized lymphocytes at the level of both the tumour cells and effector cells were demonstrable in culture supernatants of NL pretreated with TLRNA (50 or 100 microgram/4 X 10(6) cells) but not ILRNA. However, treatment of NL with either RNA resulted in the production factors that mediated tumour-specific ADCC. Cytotoxicity testing and absorption studies of the tumour cell and a control cell (LM) indicated that factors mediating ADCC and blocking at the target-cell level were specific for the tumour. Suppressor activity at the effector-cell level was not absorbed by tumour cells and represents a separate and distinct mechanism of immunosuppression. These data indicate that RNA faithfully transfers "suppressive" as well as "positive" types of immune responses that have been reported previously for lymphocytes obtained directly from tumour-bearing and tumour-immune animals.


The aim of this study was to investigate mechanisms of anti-tumour activity and necrosis induced by combinations of tumour necrosis factor alpha  and interferon gamma (IFN-gamma). In a breast cancer xenograft model, locally injected recombinant human TNF-alpha arrested growth of established tumours in the absence of overt necrosis. Macroscopic necrosis occurred when rat IFN-gamma, which had no anti-tumour activity as a single agent, was given systemically. Treatment with TNF-alpha and IFN-gamma caused focal engorgement of tumour capillaries with erythrocytes, intravascular recruitment of polymorphonuclear cells and platelet adherence to the tumour vascular endothelium 4 h after the combined treatment. This was followed by destruction of tumour vascular endothelium and both necrosis and apoptosis of tumour cells. Concomitant with these changes, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the increase of stromal (murine) mRNA levels for TNF-alpha, TNF receptor 55 kDa, TNF receptor 75 kDa, intracellular adhesion molecule 1, vascular cell adhesion molecule 1, P-selectin and interleukin 6 (IL-6). Thus, the effect of the combined TNF-alpha and IFN-gamma therapy involved the selective destruction of the tumour vasculature, death of tumour cells and increased expression of a series of stromal cytokines, cytokine receptors and adhesion molecules, which could be implicated in the observed events.


The effect of charge modification of photoimmunoconjugates (PICs) on their biodistribution in a xenograft model of ovarian cancer was investigated. Chlorin(e6)c(e6) was attached site specifically to the F(ab')2 fragment of the murine monoclonal antibody OC125, directed against human ovarian cancer cells, via poly-1-lysine linkers carrying cationic or anionic charges. Preservation of immunoreactivity was checked by enzyme-linked immunosorbent assay (ELISA). PICs were radiolabelled with 125I and compared with non-specific rabbit IgG PICs after intraperitoneal (i.p.) injection into nude mice. Samples were taken from normal organs and tumour at 3 h and 24 h. Tumour to normal 125I ratios showed that the cationic OC125F(ab')2 PIC had the highest tumour selectivity. Ratios for c(e6) were uniformly higher than for 125I, indicating that c(e6) became separated from 125I. OC125F(ab')2 gave highest tissue values of 125I, followed by cationic OC125F(ab')2 PIC; other species were much lower. The amounts of c(e6) delivered per gram of tumour were much higher for cationic OC125F(ab')2 PIC than for other species. The results indicate that cationic charge stimulates the endocytosis and lysosomal degradation of the OC125F(ab')2-pl-c(e6) that has bound to the i.p. tumour. Positively charged PICs may have applications in the i.p. photoimmunotherapy of minimal residual ovarian cancer.


In 14 consecutive patients with recurrent melanoma of the lower limb a total of 35 biopsies were taken at the end of perfusion treatment to assess melphalan tissue concentrations in tumour, skin/subcutis and muscle tissue. In tumour tissue (n = 12) the mean melphalan concentration was 6.8 micrograms g-1, which was significantly higher than that of healthy skin/subcutis , but equal to that of muscle tissue . The correlation between melphalan concentration in the tissues and the concentration in the perfusate was studied. The latter was assessed in the form of melphalan peak concentration and the area under the curve (AUC0-->60) of the melphalan concentration-time curve. Tumour concentration proved to be correlated linearly with AUC0-->60  and muscle concentration with melphalan peak concentration . There was no relation between skin/subcutis concentrations and the perfusate parameters. Further research is warranted to study the relationship between melphalan tissue concentration, tumour response and regional toxicity.


Neither radiation alone (375 to 1500 rad) nor5-fluorouracil (FU) alone (50-250 mg/kg) is sufficient to prevent an increase in the volume of the solid tumour model hepatoma 3924A. However, as little as 750 rad with 100 mg/kg FU can reduce the tumour below the volume at the time of treatment for as long as 14 days. A series of combined FU and radiation doses given every 11 days should then result in successively smaller tumour volumes until the tumour is eradicated. Changes in tumour volume were analysed by two different methods: (1) tumours in each treatment mode were grouped together and the average response to treatment determined, and (2) tumour volume changes in individual tumours were analyzed utilizing the chi2 technique, which fits the logarithmic tumour volume change with time to polynomials. This two-directional method of analysis has the advantage of permitting both an overview of the main effects of treatment via the averages, and at the same time a detailed examination of the mechanism by which these effects occur through the analysis of individual response. The results suggest that, in addition to concentrating on the cellular response immediately after therapy, greater emphasis should be placed on the kinetic changes of the tumour 1-3 weeks after single or multiple modality therapy. These findings demonstrate how the sequencing of single and/or combined treatment modalities may be investigated in order to detemine how best to obtain maximum effects of treatment on different types of tumours following recovery of the host from the previous treatment series.


Here, we investigated the direct effect of increased NF-k B activity on the tumorigenicity of MCF7 ADR cells by over-expressing the NF-k B subunit relA/p65. Surprisingly, our results demonstrated that over-expression of relA determines a considerable reduction of the tumorigenic ability in nude mice as indicated by the tumour take and the median time of tumour appearance. In vitro studies also evidenced a reduced cell proliferation and the activation of the apoptotic programme after relA over-expression. Apoptosis was associated with the production of reactive oxygen species, and the cleavage of the specific substrate Poly-ADP-ribose-polymerrase. Our data indicate that there is no general role for NF-k B in the regulation of apoptosis and tumorigenicity. In fact, even though inhibiting NF-k B activity has been reported to be lethal to tumour cells, our findings clearly suggest that an over-induction of nuclear NF-k B activity may produce the same effect. © 2001 Cancer Research Campaign http://www.bjcancer.comWe previously demonstrated that bcl-2 over-expression increases the malignant behaviour of the MCF7 ADR human breast cancer cell line and enhances nuclear factor-


Investigations of solid tumours suggest that vessel occlusion may occur when increased pressure from the tumour mass is exerted on the vessel walls. Since immature vessels are frequently found in tumours and may be particularly sensitive, such occlusion may impair tumour blood flow and have a negative impact on therapeutic outcome. In order to study the effects that occlusion may have on tumour growth patterns and therapeutic response, in this paper we develop and investigate a continuum model of vascular tumour growth.λ-ω type) dictates whether the waves develop into regular or irregular spatio-temporal oscillations. Simulations of chemotherapy reveal that treatment outcome depends crucially on the underlying tumour growth dynamics. In particular, if the dynamics are oscillatory, then therapeutic efficacy is difficult to assess since the fluctuations in the size of the tumour cell population are enhanced, compared to untreated controls.By analysing a spatially uniform submodel, we identify regions of parameter space in which the combination of tumour cell proliferation and vessel occlusion give rise to sustained temporal oscillations in the tumour cell population and in the vessel density. Alternatively, if the vessels are assumed to be less prone to collapse, stable steady state solutions are observed. When spatial effects are considered, the pattern of tumour invasion depends on the dynamics of the spatially uniform submodel. If the submodel predicts a stable steady state, then steady travelling waves are observed in the full model, and the system evolves to the same stable steady state behind the invading front. When the submodel yields oscillatory behaviour, the full model produces periodic travelling waves. The stability of the waves . Employing a combination of numerical and analytical techniques, we demonstrate how the spatio-temporal dynamics of the untreated tumour may influence its response to chemotherapy.This manuscript was reviewed by Professor Zvia Agur and Professor Marek Kimmel. Experimental observations suggest that blood vessels in solid tumours may be affected by the mechanical stress exerted on them by tumour cells. Immature vessels lacking pericytes are especially likely to be structurally unstable . As a tuet al. : p <pcrit. Various imaging modalities, e.g. CT and MRI, function in this way; the tumour can only be detected wherever its density is above the "threshold of detection"  [xmax, meaning that the therapy causes the invading wave front to recede.A way of measuring the therapy's effect on tumour size is to monitor the tumour's spatial extent. Here we investigate how far the invading wave front has spread by determining the length of the interval in which the tumour cell density exceeds a threshold value, l image) . In all ptot, where ptot is defined by the Trapezoid ruleIn our simulations we observed that the therapy had a more pronounced effect on tumour burden than on the tumour's spatial extent. Therefore in what follows we only monitor the tumour burden, pi = p, xi = iΔx for i = 0, ..., n and Δx = L/n. In Figure ptot, is highest in the case of stable tumour dynamics  when the dynamics are oscillatory, the varying initial values of ptot reveal that stable/strong vessels promote tumour growth more effectively than unstable/weak ones. Experimental observations of ovarian carcinoma spheroids have yielded similar trends: fast-growing tumours, that increased exponentially in volume, exhibited only small variations in the density of their vasculature, which mainly consisted of mature vessels [η0, and the maximum rate of vessel occlusion, δ (ensuring δ/η0 > 1). Simulations with slower/faster oscillations (and others with Dv ≠ 1) yielded similar results as those presented above.where s Figure , panel As Figure , panel Cs Figure . Since o vessels . Slow-gr vessels . In Figuδ being large, corresponding to immature vessels) and that the oscillations arise from interactions between the tumour cells and the compliant blood vessels.In this paper we have developed a PDE model which describes tumour growth within a vascularised tissue where the tumour cells rely on the vasculature for their supply of oxygen. Analysis of a spatially homogeneous submodel, for which the oxygen profile is assumed to be quasi-steady, showed that the tumour cell and vessel densities either evolve to a stable equilibrium or undergo oscillations. We found that the latter is more likely to occur if the vessels are prone to occlusion  exceeded the rate of EC proliferation (η0) then the oscillatory solutions would persist. By introducing VEGF it would also be possible to include chemotaxis with vessels moving up spatial gradients of VEGF. The inclusion of chemotaxis is likely to affect the spatio-temporal patterns that emerge as a result of tumour invasion.As with any mathematical description of a biological system, modifications could be made to increase the realism of our model. For example, we could investigate the effects that the angiopoietins, Ang-1 and Ang-2, have on the developing vasculature. To do this we would need to separate the vessels into two subpopulations consisting of immature vessels (destabilised by Ang-2) and mature vessels (stabilised by Ang-1). Immature vessels may be particularly sensitive to occlusion since they typically lack a pericyte covering [Kc). We could also investigate the tumour's response to therapy if the drug targets both tumour cells and proliferative ECs.Regarding the way in which we have modelled chemotherapy it is also possible to amend the model to more accurately describe doxorubicin activity. For example, we could investigate the effects of changing the functional form used to model cell kill in equation (2). As found in , the modδ (which appears in equation (3)) a decreasing function of the vascular-targetting drug concentration. We anticipate that this could render the system more stable, possibly enhancing the therapy's performance.Despite the simplicity of the model, the simulations of doxorubicin administration presented in this paper still give considerable insight into how the response to chemotherapy may be strongly influenced by the underlying system growth dynamics. A possible direction for further research involves extending our model to distinguish between proliferating and quiescent tumour cells in order to determine how the dynamics are affected by cell heterogeneity. We could also extend our model to investigate the impact that vessel normalisation strategies, designed to increase the stability of the immature tumour vessels, have on systems with irregular spatio-temporal dynamics. Possible candidates for vascular-targetting agents include the anti-angiogenic drug DC101 which transiently increases pericyte recruitment, hence increasing vessel stability, oxygenation, and drug delivery . ModelliIn this paper we have developed a deterministic model of vascular tumour growth formulated as a system of reaction-diffusion equations. Using a combination of numerical methods and analytical techniques we showed how the system's spatio-temporal dynamics  are influenced by the dynamics of the associated spatially-uniform system. Depending on the interplay between the proliferating tumour cells and the collapsible vessels, we observed patterns of tumour invasion ranging from regular travelling waves to waves with highly irregular spatio-temporal structure. The latter were characterised by highly responsive, compliant vessels while the former were characterised by stiffer, less compliant vessels. Simulations of chemotherapy illustrated how treatment outcome depends on the dynamics of the untreated tumour. In particular we found that the therapeutic efficacy could be dramatically reduced by the presence of immature vessels which contribute to the emergence of oscillatory dynamics.The authors declare that they have no competing interests.λ-ω analysis, with particular support from MRO.All authors contributed to the development of the mathematical model and to the production of this manuscript. IJS performed the numerical simulations and Prof. Zvia Agur, The Institute for Medical BioMathematics (IMBM), IsraelThis paper focuses on vessel occlusion. Analysis of a mathematical model for vessel occlusion, in the form of a system of reaction-diffusion equations, shows that increased occlusion may give rise to sustained oscillations in tumor cell density and in vessel density. The paper suggests that oscillations in tumor size may compromise the efficacy of Doxorubicine chemotherapy. I would like to mention a few points which may be considered in order to improve the paper.1. The basic model deliberately simplifies tumor growth and vascularisation dynamics, and couples these simple dynamics through vessel occlusion  the authors state: "To our knowledge, this is the first model of vascularised tumor growth in which the direct interactions between the tumor cells and the vasculature lead to sustained oscillations of significant amplitude". However, I believe this statement should be eliminated. Both some of the papers cited in the present manuscript and others have already showed the same result. For example, in , we have3. Another example. On page 3 parag 2 Stamper et al write: " Analysis of the model we derive in this paper reveals that the interplay between tumor and its blood vessels may lead to strongly oscillating dynamics... if the vessels are structurally unstable". Previous models with no occlusion assumption have shown the same result. Some of them, but not all, are cited in the present manuscript .4. One of the main conclusions of the paper is that : "Simulations of the chemotherapy illustrated how treatment outcome depends on the dynamics of the untreated tumor...". I believe this is already known to any scientist involved in cancer research. In any case it has been shown in many papers. I think this sentence should be suppressed.5. I suggest that sentences such as  "We found that ...oscillations arise from the interactions between the tumor cells and the compliant blood vessels" would rather be eliminated.6. I have not found any reference to the pharmakodynamics of doxorubicine. What is the assumed drug action mechanism? Why does the model represent doxorubicine and no other chemotherapy ?Top sum up, I think that the paper will benefit from a careful editing where the main messages are spelled out consistently through the Abstract, Results, and Conclusions sections, and where the special assumptions and simplifications are recognised so that conclusions and implications are more restricted.We agree with Professor Agur that our model is not the first one of vascular tumour growth to produce oscillatory dynamics - that is why we cite the seminal work of Agur and coworkers, amongst others. The essential novelty in our paper arises from the investigation of how the interplay between the oscillations associated with the underlying time-dependent model and diffusive effects can give rise to highly irregular spatio-temporal dynamics within the tumour mass and their impact on the tumour's response to therapy.Prof. Marek Kimmel, Department of Statistics, Rice University, USAA comment concerning the catalogue of spatial oscillatory patterns. In the paper  by MarciWe thank Professor Kimmel for directing us to this interesting publication. There are some similarities with our work, in that we consider diffusion-driven instability (DDI) as a mechanism for destabilising spatially homogeneous solutions. However an important difference between our work and that of Marciniak-Czochra and Kimmel is that we also use lambda-omega analysis to investigate the stability of time-periodic model solutions to spatial perturbations. In this way we provide a possible explanation for the highly irregular spatio-temporal dynamics that characterise many vascularised tumours and show that such dynamics may have a deleterious impact on the tumour's response to chemotherapy.Appendix. In the pdf file Appendix.pdf we discuss our model parameters and estimate values for some of them. We also give additional information relating to the λ-ω analysis of our model.Click here for file


Sir,et al, concerning our article TH/2008/4030R published in Br J Cancer 99/1 July 2008.We read with great attention the letter by Eveno The main point of their letter is that murine models are not suited as evidence of an effect of bevacizumab on tumour growth, because bevacizumab fails to efficiently neutralise murine VEGF.in vitro on isolated endothelial cells, whereas ours have been performed in xenografted animals. In our conditions human VEGF, produced in very high quantities by the CAL33 human cancer cells, certainly outnumbered the murine VEGF at least inside the tumour and in its immediate environment. It is not excluded that a greater dose of bevacizumab could perhaps have been more efficient.Their experiments have been performed Anyway, the purpose of our experiments was not to test the effect of bevacizumab alone on CAL33 growth but rather its interaction with erlotinib and radiation.3, 12 days after tumour cell injection  and the effect of bevacizumab was clearly evident 4 days after the beginning of treatment and lasted until the end of the observation period, 26 days after the beginning of treatment.Another explanation of the lack of efficiency of bevacizumab given alone could result from the orthotopic model used with a very short time given to the drug to exhibit its effects  giving treatment on small tumours at a moment when tumour-driven angiogenesis is not particularly determinant. What gives credit to this explanation are the results of another recent study of our group (Moreover, many earlier preclinical studies performed on xenografted tumours (for review see et al that the present animal models are far from being a perfect representation of the clinical situation and that animals genetically modified to secrete human VEGF in place of murine VEGF model could be an improvement to the present situation.We agree with Eveno


The metal complexes Pd(MP)2.2H2O, Pt(MP)2H2O (MPH=6-mercaptopurine), Pt(AMP2.3H2O and Pd3(AMP)4Cl2(AMPH).4H2O (AMPH=thioguanine) have been isolated. They were screened for anti-tumour activity in the L-1210 lymphoid leukaemia test system in mice. All 4 show marked anti-tumour activity, the complex Pt(AMP)2.3H2O giving a T/C of 185 at the optimum dosage. However, the anti-tumour activity of the metal complexes is somewhat less than that shown by the parent purines under the same conditions.


The ascorbyl radical concentration has been observed, by means of electron spin resonance spectroscopy, in the blood and spleen of female Wistar rats carrying a Yoshida tumour. The ascorbyl radical concentration of the tumour tissue itself was studied as the tumour was developing, and as it was regressing after treatment with methylene dimethane sulphonate. Changes in the concentration of this radical may be related to host tumour reactions.


Levels of glutathione (GSH) in tumour tissue may be important in determining the clinical response to certain anticancer agents. Recent reports have suggested that D,L-buthionine-S,R-sulphoximine (BSO), a specific inhibitor of GSH synthesis, may be used to deplete tumour cell GSH and thus increase the therapeutic ratio of these agents. We have previously shown that 1-naphthol is a potential antitumour agent, and that its possible metabolite 1,4-naphthoquinone is thiol reactive and capable of redox cycling. It was therefore of interest to investigate the effect of pretreatment with BSO, on the toxicity of these agents, to tumour cells. For comparison we included three other cytotoxic agents, melphalan, helenalin and menadione, the toxicities of which are reported to be modulated by intracellular GSH. Depletion of GSH using BSO did not effect the toxicity of 1-naphthol, or 1,4-NQ but did produce slight potentiation of the cytotoxicities of menadione, helanalin and melphalan. The lack of effect of BSO on 1-naphthol and 1,4-NQ is not easily explained but if one also considers the modest potentiation of cytotoxicity+ achieved with the other agents studied, the potential use of BSO in combined chemotherapy is at best rather modest.


The relationship between the utilization of 3H-thymidine in situ  and tumour growth delay after treatment with various cytotoxic agents has been examined. It is shown that (a) it is not possible to predict tumour growth delay, or to select the most effective agent, from changes in TFI 1 day after treatment; (b) there is a good correlation between tumour growth delay and the time for recovery of TFI to the pretreatment level; (c) there is a relationship within a tumour line between the depression of TFI 4 days after treatment and growth dealy induced by the same treatment. This relationship appears to be independent of the mechanism by which the agent exerts its cytotoxic effect.


An isolated rat hindlimb perfusion model carrying xenografts of the human melanoma cell line MM96 was used to study the effects of perfusion conditions on melphalan distribution. Krebs-Henseleit buffer and Hartmann's solution containing 4.7% bovine serum albumin (BSA) or 2.8% dextran 40 were used as perfusates. Melphalan concentrations in perfusate, tumour nodules and normal tissues were measured using high-performance liquid chromatography (HPLC). Increasing the perfusion flow rates (from 4 to 8 ml min(-1)) resulted in higher tissue blood flow (determined with 51Cr-labelled microspheres) and melphalan uptake by tumour and normal tissues. The distribution of melphalan within tumour nodules and normal tissues was similar for both Krebs-Henseleit buffer and Hartmann's solution; however, tissue concentrations of melphalan were significantly higher for a perfusate containing 2.8% dextran 40 than for one containing 4.7% BSA. The melphalan concentration in the tumour was one-third of that found in the skin if the perfusate contained 4.7% BSA. In conclusion, this study has shown that a high perfusion flow enhances the delivery of melphalan into implanted tumour nodules and normal tissues, and a perfusate with low melphalan binding  is preferred for maximum uptake of drug by the tumour.


Vascular endothelial growth factor (VEGF) plays an important role in tumour angiogenesis. VEGF binds to tyrosine kinase receptors, which are expressed almost exclusively on tumour endothelium. Therefore, VEGF can be used to target toxin molecules to tumour vessels for anti-angiogenic therapy. However, recent evidence suggests that VEGF can also bind in an isoform-specific fashion to a newly identified neuropilin-1 (NP-1) receptor. NP-1 is widely expressed in normal tissue and presents a potential target for unwanted toxicity. As a consequence, we investigated whether the VEGF121 isoform, which lacks the NP-1 binding domain, could be used to target toxin polypeptides to tumour vasculature. Treatment of endothelial cells with a VEGF121–diphtheria toxin (DT385) conjugate selectively inhibited proliferating endothelial cells, whereas confluent cultures were completely resistant to the construct. In addition, VEGF121–DT385 conjugate treatment completely prevented tumour cell induced angiogenesis in vivo. Most importantly, the conjugate inhibited tumour growth in athymic mice and induced tumour-specific vascular damage. There was also no apparent toxicity associated with the treatment. Our results suggest that proliferating endothelial cells are highly sensitive to VEGF121–toxin conjugates and that the binding to NP-1 receptors is not necessary for efficient inhibition of tumour growth. © 2000 Cancer Research Campaign


The successful development of the concept of linking cell-killing agents to tumour-specific antibodies will be largely determined by the extent to which the antibodies are preferentially localized in the malignant tissue. A xenograft of human choriocarcinoma (CC3) has been established in nude mice, and the relative distribution of affinity-purified specific antibodies to human chorionic gonadotrophin has been compared with that of nonspecific antibodies from the same species. Treatment of the nonspecific antibodies with ammonium thiocyanate appeared to be important to ensure that the distributions in normal nude mice were equivalent. Specificity indices, derived from the comparative distributions of isotope activity in the tumour and lung of labelled specific and nonspecific antibodies, ranged between 1.3 and 2.0.


Results indicated that 15 mg/kg of iodoacetamide sensitized hypoxic tumour cells, as did atmospheric oxygen, and the sensitization was not detectable below this dose. Experiments with fractionated treatments suggested that the reoxygenation occurring during the treatment intervals of 24 hours might be more important in the sterilization of tumour cells than the agent.The radiosensitizing effect of iodoacetamide was studied in a C3H mouse mammary carcinoma together with its toxicity to the host. TCD


A malignant invasive fibrosarcoma in hamsters and a malignant mammary carcinoma in mice were each challenged with the broad spectrum proteinase inhibitor aprotinin (Trasylol). In both tumour systems, significant anti-tumour effects of aprotinin were observed. Variations in the site and dosage of aprotinin application were made in an attempt to improve the chemotherapeutic response.


The growth of a 3-methylcholanthrene-induced fibrosarcoma of C3H mice was inhibited by aspirin and indomethacin. While the tumour contained relatively high concentrations of PGE2-like material, that were markedly diminished by indomethacin treatment, our results did not confirm the recently proposed hypothesis that the anti-tumour effect arises from a restoration of depressed immune function. For example, mice that had completely eliminated their tumours under indomethacin administration were not immune to rechallenge. The tumour-bearing animals were not non-specifically immunodepressed, as their splenic PFC responses against SRBC were enhanced. However, while indomethacin augmented the PFC response in normal mice, this adjuvant effect was depressed in tumour-bearing animals. The spleen-cell PHA responses of tumour bearers were severely depressed, and such cells suppressed the PHA response of normal cells. Only after prolonged indomethacin treatment did animals (with comparable tumour burdens) show weak PHA responses and somewhat diminished suppressive activity. Possible alternative mechanisms, such as direct cytotoxicity, or inhibition of inflammation, phosphodiesterase activity, blood coagulation or calcium availability were not implicated (nor definitively excluded) in the anti-tumour effect.


Frequency distributions for intracapillary HbO2 saturation were determined for two murine tumour lines  and two human ovarian carcinoma xenograft lines  using a cryospectrophotometric method. The aim was to search for possible relationships between HbO2 saturation status and tumour volume, tumour pH and fraction of radiobiologically hypoxic cells. Tumour pH was measured by 31P NMR spectroscopy. Hypoxic fractions were determined from cell survival curves for tumours irradiated in vivo and assayed in vitro. Tumours in the volume range 100-4000 mm3 were studied and the majority of the vessels were found to have HbO2 saturations below 10%. The volume-dependence of the HbO2 frequency distributions differed significantly among the four tumour lines; HbO2 saturation status decreased with increasing tumour volume for the KHT, RIF-1 and MLS lines and was independent of tumour volume for the OWI line. The data indicated that the rate of decrease in HbO2 saturation status during tumour growth was related to the rate of development of necrosis. The volume-dependence of tumour pH was very similar to that of the HbO2 saturation status for all tumour lines. Significant correlations were therefore found between HbO2 saturation status and tumour pH, both within tumour lines and across the four tumour lines, reflecting that the volume-dependence of both parameters probably was a compulsory consequence of reduced oxygen supply conditions during tumour growth. Hypoxic fraction increased during tumour growth for the KHT, RIF-1 and MLS lines and was volume-independent for the OWI line, suggesting a relationship between HbO2 saturation status and hypoxic fraction within tumour lines. However, there was no correlation between these two parameters across the four tumour lines, indicating that the hypoxic fraction of a tumour is not determined only by the oxygen supply conditions; other parameters may also be important, e.g. oxygen diffusivity, rate of oxygen consumption and cell survival time under hypoxic stress.


Endogenous histamine has been shown to effect growth mechanisms in experimental mammary carcinomas via H2 membrane receptors . Both H1 and H2 binding sites are present in human mammary glands but only 75% malignant carcinomas express H2 receptors . The presence of mast cells around tumour tissue raises questions concerning the source of histamine in breast tumour tissue. While cimetidine, an H2 antagonist, has been shown to influence the presence of tumour infiltrating lymphocytes (TIL) in colorectal cancer  that was not found to be the case in breast cancer . In recent studies tumour cell proliferation, as measured by Ki-67 antibody labelling, has been seen as an additional prognostic indicator in breast cancer . We investigated the possibility that cimetidine may influence tumour proliferation by blocking the growth-promoting effects of histamine. No relationship between preoperative cimetidine administration and tumour cell proliferation was seen overall. A weak correlation was seen between tissue histamine content and mast cell count which was not influenced by cimetidine. Tumour cell proliferation correlated well with other prognostic indicators such as grade and differentiation. © 2000 Cancer Research Campaign


Systemic virus dissemination is a potential problem during local gene delivery in solid tumours. However, the kinetics and pathways of the dissemination have not been well characterised during the first 24 h after the infusion is started. To this end, we infused adenoviral vectors for luciferase or enhanced green fluorescence protein into three different tumour models in mice. During and/or after the infusion, we determined the amount of adenoviruses in the tumour, blood, and liver, and examined the transgene expression in the liver, lung, blood, and tumour. In addition, we intravenously injected tumour cells expressing luciferase and examined the biodistribution of these cells in the body. We observed transgene expression in the liver and tumour at 24 h after the infusion, but could not detect transgene expression in the blood and lung. The peak concentration of viral vectors in the plasma occurred during the intratumoral infusion. At 10 min after the infusion, few viral vectors remained in the blood and the ratio of copy numbers of adenoviruses between liver and tumour was >2 in 80% and ⩾10 in 40% of the mice. Most tumour cells injected intravenously accumulated in the lung within the first 24 h. Taken together, these data indicated that systemic virus dissemination occurred mainly during the first 10 min after the intratumoral infusion was started, and that the dissemination was due to infusion-induced convective transport of viral vectors into leaky tumour microvessels. Gene therapy represents a new paradigm to treat cancer at the genetic level . Its sucThe improvement can be achieved through increasing the specificity of either vector delivery or transgene expression in tumour cells. One way to increase the delivery specificity is to physicochemically or genetically modify viral surface proteins so that the vectors will target specific biomarkers in tumours  and luciferase, respectively. The cDNA for EGFP or luciferase was inserted into the E1 region of the adenovirus and the transgene expression was driven by the cytomegalovirus (CMV) promoter. Adenoviruses were propagated in 293 cells , harvested at 48 h after infection, and purified by the method of cesium chloride gradient centrifugation according to a standard protocol . The vir2 and 95% air. For tumour implantation in mice, the cells were removed from culture flasks with 0.25% trypsin/EDTA and rinsed twice in PBS.Three tumour cell lines  were used in the study. 4T1 is a murine mammary carcinoma; B16.F10 is a metastatic subline of B16 murine melanoma; IMR-23 is a human neuroblastoma. All tumour cells were cultured in DMEM supplemented with 10% newborn bovine serum  and 1% penicillin/streptomycin  at 37°C with 5% COμl PBS were injected subcutaneously into the right hind limb of mice. After implantation, the mice were placed on a warm pad until they woke up. All subcutaneous tumours were ready for experiments when they reached 5–8 mm in diameter. After experiments, fully anaesthetised mice were killed with cervical dislocation. The use of animals and all experimental procedures involving animals in this study had been approved by the Duke University Institutional Animal Care and Use Committee. These procedures were also consistent with the United Kingdom Co-ordinating Committee on Cancer Research (UKCCCR) Guidelines  for the implantation of 4T1 and B16.F10 tumours, respectively. Female Balb/c nude mice at the same age as Balb/c mice were ordered from the National Cancer Institute  for the implantation of IMR-23 tumour. For tumour cell implantation, mice were first anesthetised with an intraperitoneal injection (0.15 ml) of a mixture of ketamine and xylazine (80 mg ketamine and 10 mg xylazine per kg body weight). Then, one million tumour cells in 50 idelines .μl although the dose of viruses varied with experiments as indicated below. The infusion rate was chosen to be 1 μl s−1 based on a preliminary study in which we infused 50 μl of a solution of Evans blue dye or a suspension of an adenoviral vector for LacZ, AdCMVLacZ, into 4T1 tumours and observed that the distribution volume of Evans blue or β-galactosidase in tumours was the highest when the infusion rate was 1 μl s−1.Mice were anesthetised with an intraperitoneal injection (0.15 ml) of a mixture of ketamine and xylazine (80 mg ketamine and 10 mg xylazine per kg body weight). The tip of a 30-gauge needle was carefully placed near the centre of tumours by controlling the depth of needle insertion relative to the tumour size measured by a caliper. Virus suspension was infused into tumours via the needle mounted on a syringe pump . The volume of infusion was fixed at 50 8 plaque forming units (pfu)), mice were anesthetised again with the same method as described above and transgene expression in tumours was examined. For luciferase analysis, 50 μl of aqueous D-luciferin solution was injected intraperitoneally into the mice placed on a warm pad at 20 min before the examination of bioluminescence in the body. The bioluminescence was recorded by the In Vivo Imaging System (IVIS) , with an exposure time of 30 s (μm thick), using a Vibratome . Enhanced green fluorescence protein expression in the blood and sliced tissues was examined under a confocal laser-scanning microscope .At 24 h after intratumoral infusion of AdCMVLuc or AdCMVEGFP  into 4T1 tumours, blood samples (approximately 20 μl in each sample) from anesthetised mice were collected through the orbital sinus with a heparinised capillary, stored at room temperature for 10 min, and centrifuged at 10 000 g for 3 min to separate the plasma from blood cells. The plasma and cell suspension were then exposed to four cycles of thawing (37°C) and freezing (−196°C in liquid nitrogen) treatment. In all, 1 μl of plasma solution or suspension of lysed blood cells was added into 96-well plates with 70–80% confluent 293 cells. After 24 h, EGFP expression in these cells was examined under a fluorescence microscope .At predetermined time points  after starting the infusion of AdCMVEGFP (3.0 × 108 pfu) into 4T1 tumors, mice were killed with cervical dislocation and the liver and tumours were immediately harvested, frozen in liquid nitrogen, and stored at −82°C. For viral DNA analysis, the tissue samples were immersed in liquid nitrogen and ground into fine tissue powder in a mortar. Viral DNA was isolated from the tissue powder using a DNeasy Tissue kit . The DNeasy mini column was eluted twice with 200 μl of Tris buffer  to obtain viral DNA solution, in which Ad E4 copy number was measured with the Smart Cycler System . In the real-time PCR analysis, the primers and probes were purchased from Sigma-Genosis . The forward primer sequence for Ad E4 region was: 5′-TGACACGCATACTCGGAGCTA-3′: 34 885–34 905; the reverse primer sequence was: 5′-TTTGAGCAGCACCTTGCATT-3′: 34 977–34 958; the fluorogenic probe sequence was: [DFAM]CGCCGCCCATGCAACAAGCTT[DBH1]: 34 930–34 951 (μl reaction mixture containing 0.15 μl of AmpliTaq Gold DNA polymerase (5 U μl−1) , 300 nM each primer, and 200 nM fluorogenic probe. The amplification consisted of an initial heating at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 45 s. The standard curve was generated by using serial dilutions of pFG140 plasmid DNA  in the genomic liver DNA solution.At 10 min after the infusion of AdCMVEGFP (3.0 × 10μl). At 24 h after infusion, strong bioluminescence, an indication of luciferase expression, was observed in the liver and tumour in all animal models (see To demonstrate the systemic dissemination of viral vectors, AdCMVLuc suspension was infused into two murine tumour models, 4T1 and B16.F10, respectively, and one human tumour xenograft, IMR-23. Bleeding was observed in many tumours immediately after the removal of infusion needle but the amount of bleeding was much less than the infused volume (i.e. 50 dels see . The bioin vitro (see in vitro (see The plasma from blood samples at different time points, both during and after the intratumoral infusion of AdCMVEGFP, contained the viral vectors that could transfect 293 cells itro see . Qualitaitro see , indicatTo further investigate the role of tumour or blood-borne cells in virus dissemination, we performed two additional experiments. Firstly, we infused luciferase-expressing cells into the tail vein of mice, and observed that nearly all infused cells accumulated in the lung . SecondlTo quantitatively understand the significance of systemic adenovirus dissemination at early time points, we quantified the copy numbers of adenoviruses in liver and tumour tissues at 10 min after the intratumoral infusion of AdCMVEGFP. We chose this time point for three reasons. Firstly, data in the literature show that adenoviruses have a plasma half-life of ∼2 min (The ratio of the copy numbers of adenoviruses between liver and tumour is shown in We observed the systemic virus dissemination in all three animal models after intratumoral infusion of adenoviral vectors. In 4T1 tumours, the dissemination occurred mainly during the first 10 min after the infusion was started. The amount of viruses disseminated into the liver could be one order of magnitude higher than that retained in the tumour. The blood and lung in mice contained few transfected cells after the intratumoral infusion. However, tumour cells injected intravenously into the mice accumulated mainly in the lung. These observations in combination with data in the literature allowed us to determine the mechanism of systemic virus dissemination during the first 24 h after the intratumoral infusion was started.Potentially, the mechanisms of dissemination include cell-mediated transport and diffusion and convection of viral vectors from the interstitial space into the lumen of tumour microvessels. We have showed previously that the diffusion process is too slow to cause the virus dissemination after intratumoral infusion (The cell-mediated transport was likely to be insignificant as well. The reasons are as follows. First, the amount of adenoviral vectors associated with cells in blood samples shown in Convection is in general negligible in the centre of solid tumours, due to uniformly elevated interstitial fluid pressure and intrinsic leakiness of tumour microvessels . ConvectTaken together, the virus dissemination occurred after the adenoviral vectors entered the system circulation via infusion-induced convective transport. The entrances were the leaky microvessels that were either intrinsically hyperpermeable, with pores in the microvessel wall being larger than adenoviruses  or damagThe ratio of copy numbers of adenoviruses between liver and tumour shown in Systemic virus dissemination after intratumoral infusion has been investigated in previous studies (In a clinical study, the amount of adenoviral DNA in the blood at 30 min after the intratumoral infusion has varied between 0.007 and 0.129% of that infused into tumours  and 5.In summary, we demonstrated that the virus dissemination occurred during the first few minutes after the intratumoral infusion was started. It was due to infusion-induced convective transport of viral vectors into leaky tumour microvessels. In future studies, we will investigate effects of infusion rate and pressure as well as structures of tumour tissues on virus dissemination. The findings in this study suggest that it is desirable to develop polymeric delivery systems that can increase the concentration of adenoviruses in solid tumours and simultaneously reduce the amount of disseminated adenoviruses in normal tissues, particularly in the liver, thereby improving the efficacy/toxicity ratio in cancer gene therapy.


The following funding information was omitted. MC also received Fondazione Cariplo grant 2005-1166 (to S.Canevari).


The nonangiogenic lung tumour is characterized by neoplastic cells co-opting the pre-existent vasculature and filling the alveoli space. 3-Dimensional reconstruction of the tumour reveals that this particular tumour progresses without neovascularization and there is no major destruction of the lung's architectural integrity. In recent years, the role of angiogenesis in neoplastic growth has become controversial. Initially, it was thought that the formation of new capillaries (neovascularization) usually mediated by angiogenic molecules released by tumour cells and activated macrophages was essential for all tumour growth  reconstructions to demonstrate the distinct differences in vascularity and morphology between the nonangiogenic and angiogenic lung tumours. We also show that in nonangiogenic tumours the integrity of the lung architecture is retained while these alveolar entrapped neoplastic cells continue to thrive without producing new vessels of their own.μm thick sections of paraffin-embedded tissue were cut from each case and mounted on slides coated with poly-L-lysine.Our model for this reconstruction is non-small cell carcinoma of the lung. Both angiogenic and nonangiogenic tissues are embedded in paraffin. The region of the tissue used for the study is 2 cm in depth. In all, 200 5 Primary non-small cell lung cancer tissues (angiogenic and nonangiogenic) were obtained with informed consent from two patients who underwent radical surgical resection. The normal tissue was obtained from a patient with a lung secondary who also underwent radical resection. This research project was approved by the local ethical committee. The diagnosis was established on routine formalin-fixed paraffin-embedded material. One section was stained with haematoxylin and eosin to verify the presence of viable tumour; another was immunostained for CD34 to assess the vascular pattern as previously described.Antigen retrieval is as previously described by Blood vessels and cytoskeleton on the dewaxed sections were identified by simultaneous immunostaining with 1 : 500 dilution of anti CD34 antibody QBEnd/10  and 1 : 25 dilution of a pan-cytokeratin rabbit polyclonal antiserum . The primary antibody staining was allowed to incubate for 1 h, followed by a 5 min wash with TBS. Immunodetection was carried out for blood vessels and cytoskeleton by another simultaneous 1 h incubation with 1 : 200 dilution of both Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 568 goat anti-mouse . The immunostained sections were washed in TBS for 5 min and then mounted in antifade Dako Fluorescent mounting medium  containing a 1 : 500 dilution of 4′,6-diamidine-2′-phenylindole dihydrochloride DAPI .Specific areas on immunostained sections were examined with the × 10 Plan fluor objective lens of a Nikon Eclipse E600 Fluorescence microscope and photographed by an interfaced Zeiss Axiocam (Germany). Photographed images were then captured by Axiovision software in an interfaced computer. The emitting fluorescence signals were selected, respectively, by a group of filters, Dappi, FITC, Tx Red and exhibited with a resolution of 1300 by 1030 pixels of Red Green Blue (RGB). Acquired images were then batch converted from Axiovision to Photoshop where all three colours of individual images were superimposed and consecutive slide images were then stacked on top of each other for 3D reconstruction/restoration of the spatial orientation of section images. The stacked image was then imported to Imaris (Bitplane) software for the 3D rendering of the images.As we expected, it was evident from the 2D picture and 3D rendering  that theFor this experiment, we chose to use the fluorescence staining technique because its advantage over regular optical microscopy is the convenience of selectively observing the structure of the nuclei (blue), vasculature (red) and cytokeratin (green) staining individually. With this technique, there is also the option to superimpose any of the image staining upon each other. This way, we were able to trace specific regions and easily construct the 2D  and 3D mWhat we have done with this study is to provide morphological evidence that a nonangiogenic tumour phenotype does exist. This then raises the question of whether this nonangiogenic phenotype has relevant biological differences to the angiogenic form. If this were the case, it would have major implications with respect to any antiangiogenic treatments.Studies in mice have shown that not all experimental metastases respond to antiangiogenic agents . ClinicaHaving established the existence of a putative nonangiogenic tumour phenotype, we propose that a possible explanation for this phenomena could be as a result of the evolution of tumour genes via clonal selection. Owing to hypoxic events that frequently occur during cancer cell progression, genes such as those governing efficient regulation of oxygen homeostasis could be properties clonally coselected for by evolving cancer cells . This su


Treatment of cell lines in vitro with HGS-ETR1 enhanced the cytotoxicity of chemotherapeutic agents  even in tumour cell lines that were not sensitive to HGS-ETR1 alone. In vivo administration of HGS-ETR1 resulted in rapid tumour regression or repression of tumour growth in pre-established colon, non-small-cell lung, and renal tumours in xenograft models. Combination of HGS-ETR1 with chemotherapeutic agents  in three independent colon cancer xenograft models resulted in an enhanced antitumour efficacy compared to either agent alone. Pharmacokinetic studies in the mouse following intravenous injection showed that HGS-ETR1 serum concentrations were biphasic with a terminal half-life of 6.9–8.7 days and a steady-state volume of distribution of approximately 60 ml kg−1. Clearance was 3.6–5.7 ml−1 day−1 kg−1. These data suggest that HGS-ETR1 is a specific and potent antitumour agent with favourable pharmacokinetic characteristics and the potential to provide therapeutic benefit for a broad range of human malignancies.Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of tumour cells through activation of TRAIL-R1 and TRAIL-R2 death signalling receptors. Here, we describe the characterisation and activity of HGS-ETR1, the first fully human, agonistic TRAIL-R1 mAb that is being developed as an antitumour therapeutic agent. HGS-ETR1 showed specific binding to TRAIL-R1 receptor. HGS-ETR1 reduced the viability of multiple types of tumour cells  Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), also known as Apo2L, is a member of the TNF ligand superfamily .c and Smac/DIABLO from the mitochondria and subsequent activation of caspase 9 , consisting of death receptors, adaptor proteins, and procaspase 8, which leads to processing and activation of procaspase 8 by an autocatalytic mechanism , a fully human agonistic monoclonal antibody with high affinity and specificity for TRAIL-R1. HGS-ETR1 induced cell death in tumour cell lines and this killing was mediated through the activation of both extrinsic and intrinsic death signalling pathways. HGS-ETR1 was shown to have a long half-life Tumour cell lines Colo205, HCT116, H460, H2122, ST486, SW480, RL95-2, SU.86.86, ES2, A498, WM793, and SNU398  overnight at 4°C. Three or four rounds of panning selections were performed using the scFv human antibody phage library with approximately 1.4 × 1010 individual recombinants (Phage display was used to isolate anti-TRAIL-R1 antibodies. A large nonimmunised human scFv (single chain variable fragment) phage display library was used for selection as described previously (M) were added. After incubation for 2 h at room temperature, the wells were washed and bound HGS-ETR1 antibody was detected using peroxidase-conjugated goat anti-human IgG Fab and tetramethylbenzidine (TMB) horseradish peroxidase (HRP) substrate solution  according to the manufacturer's instructions.To determine direct binding of fully human IgG1 HGS-ETR1 to TRAIL-R1, TRAIL-receptor extracellular domain fusion proteins  were immobilised on 96-well plates, and various concentrations of HGS-ETR1 (0.9–66.7 n3 and 0.1% BSA) and resuspended at 106 cells per 100 μl. Cell surface expression of TRAIL receptors was determined using 10 μg ml−1  of HGS-ETR1 or commercial mouse anti-TRAIL-R1 antibodies . Matched isotype control human IgG (IC mAb) or commercial mouse IgG (eBiosciences) monoclonal antibodies were used as negative controls. Cells were incubated with antibodies for 20 min at room temperature, washed, resuspended in 200 μl FACS buffer containing 0.5 μg ml−1 propidium iodide and analysed on a FACScan . To determine specificity of HGS-ETR1 binding, FACS analysis was performed in the presence of 5 μg ml−1 of soluble TRAIL receptor-Fc fusion proteins .Adherent cell lines were detached from flasks using EnzFree buffer , washed with FACS buffer .Cell viability was determined using Cell Titer-Glo . Cells were plated in 96-well white polystyrene opaque plates  and incubated overnight at 37°C. Indicated concentrations of HGS-ETR1 antibody or controls were added. In combination studies, chemotherapeutic agents were also added at the indicated concentrations with either control antibody or HGS-ETR1. After 48 h incubation, cell viability was measured according to the manufacturer's instructions. In brief, 100 4 cells well−1 and cultured overnight. Cells were treated with HGS-ETR1 or controls for the indicated times at 37°C and caspase activity was measured using a tagged caspase substrate (rhodamine-labeled DEVD peptide). The assay was performed according to the manufacturer's instructions  and the release of the fluorogenic moiety by activated caspases was measured by reading plates at 405 nm using a fluorometric plate reader. Treatments were performed in triplicate and the average and standard deviation determined and plotted.Cells were plated in black-walled 96-well plates (Costar) at 1 × 106) were plated overnight in a 150-mm cell culture plate and then treated with the indicated concentrations of HGS-ETR1 or control antibody for 4 h at 37°C in a cell culture incubator. Cells were then scraped in ice-cold PBS, centrifuged and cell pellets lysed with 1% NP40 lysis buffer . The protein concentration of the lysates was determined by BCA method  and normalised with lysis buffer. The proteins were separated using Tris-glycine polyacrylamide SDS gel electrophoresis and transferred to nitrocellulose membranes . The filters were incubated with antibodies that recognise the pro and cleaved forms of the apoptotic proteins PARP , caspase 3, caspase 8, caspase 9 , Bid , and actin . The bands corresponding to specific proteins were detected by HRP-conjugated secondary antibodies and enhanced chemiluminescence .Cells (2 × 107 tumour cells were injected subcutaneously (s.c.) in the lower right flank of the mouse . Prior to any treatments, tumours were grown to ∼100 mm3 and 8–10 mice per group were used for each treatment group. In the single-agent models, HGS-ETR1 or isotype control antibody was administered to the animals intravenously (i.v.) via tail vein in a dose/weight-matched fashion on indicated days. Tumours were established as above for the Colo205 and HCT116 combination treatment models. In the Colo205 combination model, 5FU mini-pumps were inserted on day 7. Each 0.5 ml h−1 mini-osmotic pump  was filled with sterile 5FU solution  for a 1.25 mg kg−1 h−1 flow rate. Mice were anesthetised with inhaled isoflurane, skin was sterilised and the mini-osmotic pumps containing 5FU were inserted s.c. in a pocket formed dorsally under the skin of the mouse. Skin was closed with two 9 mm wound clips. Wound clips were removed after 14 days. Continuous infusion of 5FU was given from days 7–21. Isotype control antibody (10 mg kg−1) was administered i.v. on days 7, 10, 12, 14, 17, 19, 21, 24, 26, 28, and 31. HGS-ETR1 (10 mg kg−1) was administered i.v. on days 14, 17, 19, 21, 24, 26, 28, and 31 either alone or in animals treated with 5FU. One group of animals was untreated. In the HCT116 combination treatment model, HGS-ETR1 or isotype control antibody (10 mg kg−1) was administered i.v. on days 11, 17, and 24. Irinotecan was administered i.p. at a dose of 8 mg kg−1 on days 11, 15, 17, 21, 24, and 28. For the SW480 combination treatment de novo model, male Swiss athymic mice (Taconic), 6–8 weeks of age and 20–25 g average body weight, were used and 1 × 107 SW480 cells were injected per site, six mice per group on day 0. On day 1, a loading dose of HGS-ETR1 (20 mg kg−1) was administered i.v. Topotecan was administered i.p. at a dose of 0.3 mg kg−1 on days 1–3. Subsequently, HGS-ETR1 (10 mg kg−1) and topotecan (0.3 mg kg−1) were administered i.p. on days 4, 7, 10, 13, and 16. One group of mice was injected with vehicle control. For all models, tumour size on two axes was measured with digital calipers at 3–4-day intervals. The values were transformed into tumour volumes using the following formula: tumour volume=0.5 × width2 × length. Unpaired Student t-tests were used to evaluate the significance of differential tumour growth. The difference was considered significant when P was <0.05.For pre-established xenograft tumour models , female Swiss athymic mice , 7–8 weeks of age and 20 g average body weight, were used. All experiments were performed in accordance with the current guidelines established by UKCCR and the Institutional Animal Care and Use Committee at Human Genome Sciences Inc. On day 0, 1 × 10−1 HGS-ETR1. Four mice were injected for each time point. Mice were killed and blood was collected at 5 min, 3 h, 8 h, 1, 2, 3, 4, 7, and 10 days postinjection. Sera prepared from the blood samples were stored at −80°C until analysed by ELISA. For the ELISA assays, a Maxisorb plate  was coated with a TRAIL-R1 fusion protein solution (2 μg ml−1 in PBS), and blocked with a PBS solution containing 10% goat serum. The sera samples to be tested for HGS-ETR1 levels were diluted 2000-fold (0.05%) in a PBS solution containing 0.05% Tween-20. HGS-ETR1 standards and positive controls were made in a PBS solution containing control 0.05% BALB/c serum. Diluted samples, standards, and positive controls were incubated on the plate for 2 h at room temperature. The plate was washed six times using a PBS solution containing 0.1% Tween-20 and incubated for 2 h with a PBS solution of 10% goat sera containing HRP-labelled anti-human λIgG antibody (1 : 80 000). The plate was washed once more prior to the addition of TMB HRP substrate. After a 15-min development time, the HRP activity was terminated using dilute sulphuric acid and the relative absorbance at 450 nm was measured using a Spectromax ELISA  plate reader. The concentration of the HGS-ETR1 in the sera samples was extrapolated from the HGS-ETR1 standard curve. The limit of quantitation of the assay is 5 ng ml−1 in 100% serum. To obtain pharmacokinetic parameters, serum concentration data were fitted with a two-compartment i.v. bolus model with 1/2 weighting using the software package WinNonlin . Data from all mice in each dose group were fit together to obtain a pooled estimate of the PK parameters.Male BALB/c mice  were obtained from Ace Animals . Mice were injected i.v. via tail vein with 10 or 20 mg kg1 and further characterised for agonist activity on multiple tumour cell types and antitumour activity in xenograft models. HGS-ETR1 was selected as the lead candidate based on its potent in vitro and in vivo antitumour effects. Binding of the fully human IgG1 form of HGS-ETR1 to the extracellular domain of human TRAIL-receptor proteins immobilised on 96-well plates was characterised using an ELISA format. Specific binding of HGS-ETR1 to the TRAIL-R1 protein but not to other TRAIL receptors was observed . Several top candidates were converted to IgGobserved . The speobserved . AgonistHGS-ETR1 .HGS-ETR1 induction of cell killing and mechanism of action were analysed in multiple tumour cell lines , representing various tumour types . All of To more fully characterise the signalling pathways used by HGS-ETR1, activation of intracellular apoptotic signalling molecules was determined by Western analysis in three cell lines. Dose-dependent activation of caspases, as determined by a decrease in procaspase 8 levels and cleavage of caspase 9 and caspase 3, was observed after a 4-h treatment of ST486 and SW480 cell lines with HGS-ETR1 . HGS-ETRin vitro was analysed in HGS-ETR1-sensitive  and -insensitive  tumour cell lines. Cells were treated with various concentrations of control antibody or HGS-ETR1 either alone or in combination with camptothecin (HCT116), cisplatin (H460), carboplatin (ES2), or 5-fluorouracil (TTn) and the cell viability was determined after 48 h , colon (Colo205), and renal (A498) tumours in xenograft models in athymic nude mice. Tumours were pre-established s.c. to a volume of approximately 100 mm3 before initiation of antibody treatments. Three weekly treatments of HGS-ETR1 at a concentration of 2.5 or 10 mg kg−1 showed strong single-agent antitumour activity in the H2122 NSCLC xenograft model, with significant tumour regression observed after a single injection of HGS-ETR1 (P<0.0001). Tumour volume was reduced approximately 50% by day 10, 4 days after the first injection of HGS-ETR1 and by day 25, there was a 97% reduction in tumour volume for the 10 mg kg−1 dose of HGS-ETR1. In addition, tumour regrowth was suppressed for several weeks after termination of HGS-ETR1 treatment (data not shown). HGS-ETR1 also induced tumour regression in colon and renal xenograft tumour models. Treatment with a 10 mg kg−1 dose of HGS-ETR1, every other day or once a week, respectively, dramatically reduced tumour volumes in Colo205 colon (P<0.0001 at day 21) and A498 renal (P<0.0001 at day 28) xenograft tumour models compared to the isotype control antibody , HGS-ETR1 alone , or combination of 5FU and HGS-ETR1, and compared to no treatment or treatment with isotype control mAb . Tumour size in nontreated animals was not significantly different from isotype control mAb treated. Both 5FU alone (P<0.001) and HGS-ETR1 alone (P<0.001) had a significant cytostatic effect on tumour growth compared to control. For the HGS-ETR1+5FU combination treatment group, mice were pretreated with 5FU for 7 days before treatment with HGS-ETR1 and resulted in an immediate decrease in tumour volume evident at day 17. The overall effect on tumour volume after combining HGS-ETR1 and 5FU was significantly different when compared to either agent alone (P<0.001).The ability of HGS-ETR1 to enhance the t models . HGS-ETRt models . Tumours3 were established and animals were treated with irinotecan , HGS-ETR1 , or isotype control antibody . At day 31, treatment with irinotecan alone resulted in a 38.8% reduction in tumour growth compared to control antibody (P<0.0113). HGS-ETR1 treatment showed a 20.1% reduction in tumour volume, which was not significantly different from that of the control antibody group (P<0.2248). The combination of HGS-ETR1 plus irinotecan resulted in a significant reduction of tumour growth (59.7%) when compared with control antibody (P<0.0007), irinotecan alone (P<0.0103), or HGS-ETR1 alone (P<0.007).The effect of combination of HGS-ETR1 with irinotecan was examined in pre-existing HCT116 colon tumours . Tumoursde novo SW480 colon tumour cell model (−1) was administered i.v. Topotecan (0.3 mg kg−1) was administered i.p. on days 1, 2, and 3. Topotecan and HGS-ETR1 (10 mg kg−1) alone or in combination were administered i.p. on days 4, 7, 10, 13, and 16. At day 28, when compared to vehicle control, topotecan alone did not show a significant effect , but HGS-ETR1 treatment alone , or coadministration of HGS-ETR1 and topotecan , significantly inhibited the growth of SW480 tumours. The reduction of tumour growth by the combination of HGS-ETR1 plus topotecan was significantly greater than topotecan alone (P<0.0194); however, significance was not reached when the combination was compared to HGS-ETR1 alone (P<0.0907).The effect of HGS-ETR1 and topotecan alone or in combination was tested in a ll model . On day −1. Serum concentrations of HGS-ETR1 were determined by ELISA  of HGS-ETR1 in mice is 6.9–8.7 days, the clearance is 4.81–5.66 ml day−1 kg−1, and the steady-state volume of distribution (Vss) is 55.3–59.0 ml kg−1. The Cmax and AUC are approximately proportional to dose. The tβ1/2, , CL, Vi, and Vss are not significantly different between dose groups, indicating that the pharmacokinetics of HGS-ETR1 were linear over the limited range of doses tested.The pharmacokinetics of HGS-ETR1 was evaluated in Balb/c mice following a single i.v. injection of 10 or 20 mg kgby ELISA . The solEngagement of cell surface death receptors by TRAIL is the initial step in the induction of intracellular death signalling pathways and apoptosis . Mouse aAlthough TRAIL-R1 cell surface expression was required for the cytotoxic activity of HGS-ETR1, TRAIL-R1 expression levels did not predict the level of sensitivity. In general, a higher expression of TRAIL-R1 was seen in the most sensitive cell types; however, we found that there are cell lines in which there is a divergence between receptor level and HGS-ETR1 activity. For example, some cells with lower levels of TRAIL-R1 (RL95-1) were eliminated to a greater extent than cells with higher TRAIL-R1 expression (H460 or TTn). Additionally, one cell line (SNU398) with strong TRAIL-R1 cell surface expression staining was completely resistant to HGS-ETR1. Similar divergent responses in cell viability and cell surface TRAIL death receptor levels were demonstrated for TRAIL . The levIn vivo, HGS-ETR1 had potent antitumour activity in pre-established colon, lung, and kidney xenograft cancer models. In these experiments, HGS-ETR1 was administered at 2- or 7-day intervals; given the half-life of HGS-ETR1 in mice (∼5–7 days), we would expect HGS-ETR1 to accumulate using these schedules. The tumour regression observed in these models demonstrated that these treatments were pharmacodynamically effective. HGS-ETR1 was highly active in the H2122 NSCLC model and induced a rapid decrease in tumour volume after a single injection of HGS-ETR1 (2.5 or 10 mg kg−1), eliciting significant tumour regression. Tumour regrowth was repressed for 20 days after the last HGS-ETR1 treatment, suggesting that the effects of HGS-ETR1 on tumour cell growth can extend beyond the treatment phase. The lack of tumour regrowth could be attributed to several factors including activity of the residual HGS-ETR1, and the significant loss of tumour cell number, tumour burden, and tumour vasculature during the initial tumour regression. HGS-ETR1 alone, at a concentration of 10 mg kg−1, also dramatically reduced tumour volume in the Colo205 colon and A498 renal models demonstrating that HGS-ETR1 had strong antitumour activity in multiple, diverse tumour types.in vitro and in vivo. Previous studies have shown that chemotherapeutic agents enhance TRAIL-induced cell killing of various tumour cells in vitro and in vivo , substantial enhancement of carboplatin- or 5FU-induced cytotoxicity was observed after cotreatment with HGS-ETR1, even in the absence of detectable activity of HGS-ETR1 alone, demonstrating a synergistic interaction between HGS-ETR1 and these chemotherapeutic agents. In vivo, enhanced efficacy of combination treatments of HGS-ETR1 with three different chemotherapy agents was demonstrated in three separate colon xenograft tumour models. In the Colo205 model, 5FU was given for 7 days via infusion, prior to HGS-ETR1 treatment to emulate a current therapeutic delivery mode for 5FU in colon cancer. Combination of HGS-ETR1 treatment with 5FU pretreatment was effective in inducing a pronounced tumour regression, suggesting that combination of chemotherapy and HGS-ETR1 do not have to be given concurrently for efficacy. Interestingly, this regression occurred immediately after injection of HGS-ETR1, suggesting that the antitumour action of the mAb to induce apoptosis and reduce tumour volume is rapid. Various mechanisms have been proposed for the enhanced cell death observed by combination of chemotherapy with TRAIL, including upregulation of TRAIL-R1 and TRAIL-R2 , although it is restricted to a space less than the total extracellular fluid volume of 170 ml kg−1 (The pharmacokinetic profile of HGS-ETR1 indicated that the half-life of HGS-ETR1 was 6.9–8.7 days. For comparison, the reported half-life of the TRAIL ligand that binds both TRAIL-R1 and TRAIL-R2 is 3.6 min in mice  and the  ml kg−1 .in vitro in a broad range of human tumour cell types via activation of both intrinsic and extrinsic signalling cascades. HGS-ETR1 had an effective pharmacokinetic half-life, and induced rapid tumour regression in multiple xenograft tumour models alone and in combination with chemotherapeutic agents. These data indicate that HGS-ETR1 has significant potential as a cancer therapeutic agent. HGS-ETR1 is currently being evaluated in Phase I/II clinical trials in patients with advanced solid or haematological tumours.Overall, we have shown that HGS-ETR1, the first reported fully human agonistic TRAIL-R1 mAb, specifically and exclusively bound to TRAIL-R1, and potently induced apoptosis


The characterisation of a new murine B cell lymphoma, A31, is described. Histopathological examination of passaged tumour indicates that initial infiltration occurs in the spleen, lymph nodes, Peyer's patches and liver, while in the terminal phase the bone marrow, gonads and occasionally the central nervous system become involved. The terminal spread is coincidental with the leukaemic phase in the tumour. The tumour cells show typical B cell characteristics in vitro. These include surface immunoglobulin (Ig) of mu, kappa isotype, surface Ia, Thy-1 negativity and an increased uptake of tritiated thymidine following incubation with lipopolysaccharide. A31 cells secrete low levels of IgM into the tissue culture fluid. Short-term culture produced only 100 ng IgM per 10(7) cells over 8 h and no tumour-associated monoclonal band could be detected in the serum of tumour-bearing mice. Chromosomal karyotypes of A31 cells gave model numbers 2n=40 normal, and 2n=41, with partial trisomy of chromosome 2, and trisomy of 17. There was loss of a chromosome 6 and the Y chromosome, together with the translocation of part of an 11 to one of the two unidentified marker chromosomes. The responses of lymphoma-bearing mice to therapeutic levels of cyclophosphamide and vincristine sulphate and also to whole body X-radiation are illustrated. This tumour may help in unravelling the complex biology of B cell lymphoma and because of its low level of Ig secretion, be of particular value in experimental immunotherapy.


Temporary reduction in blood-flow within tumour blood vessels can reduce oxygen supply leading to transient perfusion-limited hypoxia. Consequent selection of cells with mutations and reduced radiosensitivity can lead to disease progression and treatment-resistance. In the present study, we investigated whether heterogeneity of labelling after thymidine analogue administration is related to perfusion variations, and if so, could it be quantified and used as a perfusion indicator. Perfusion in murine RIF1 tumours was reduced by hydralazine or increased by nicotinamide and the mice subsequently injected with IdUrd. Tumours were halved for analysis by both flow cytometry and immunohistochemistry. Tumour sections were stained for vasculature and IdUrd. Each blood vessel was scored for the density of IdUrd-labelled cells surrounding it, using a semi-quantitative scoring system. Flow cytometry showed that the IdUrd labelling index and intensity decreased by approximately 50% after hydralazine. In tumour sections of control animals, 2.9% of vessels showed no IdUrd label. In contrast, after hydralazine almost 50% of vessels had no surrounding IdUrd labelling, whereas after nicotinamide there were fewer vessels with low labelling and a higher median score. In conclusion, changes of tumour perfusion by pharmacological agents is reflected in changes in tumour-cell labelling by the thymidine analogue IdUrd, suggesting that IdUrd labelling could be used to indicate perfusion in individual vessels in human tumours. © 2000 Cancer Research Campaign


These sera did not, however, block the cytotoxicity of lymph node cells from tumour immune rats for cells of the immunizing tumour. In addition, the tumour specific rejection antigens and embryonic antigens have been shown to have dissimilar locations in tumour cells.The tumour specific antigens and tumour associated embryonic antigens expressed on chemically induced rat hepatomata and sarcomata have been shown to be different by means of blocking antibody studies. Serum from multiparous female rats contained blocking antibody which protected both tumour and embryo cells from the