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Hyperoxia increases the uptake of 5-fluorouracil in mammary tumors independently of changes in interstitial fluid pressure and tumor stroma

Hyperoxia increases the uptake of 5-fluorouracil in mammary tumors independently of changes in... Background: Hypoxia is associated with increased resistance to chemo- and radiation-therapy. Hyperoxic treatment (hyperbaric oxygen) has previously been shown to potentiate the effect of some forms of chemotherapy, and this has been ascribed to enhanced cytotoxicity or neovascularisation. The aim of this study was to elucidate whether hyperoxia also enhances any actual uptake of 5FU (5-fluorouracil) into the tumor tissue and if this can be explained by changes in the interstitium and extracellular matrix. Methods: One group of tumor bearing rats was exposed to repeated hyperbaric oxygen (HBO) treatment (2 bar, pO = 2 bar, 4 exposures à 90 min), whereas one group was exposed to one single identical HBO treatment. Animals housed under normal atmosphere (1 bar, pO = 0.2 bar) served as controls. Three doses of 5FU were tested for dose response. Uptake of [ H]-5FU in the tumor was assessed, with special reference to factors that might have contributed, such as interstitial fluid pressure (P ), collagen content, oxygen stress (measured as malondialdehyd levels), if lymphatics and transcapillary transport in the tumors. Results: The uptake of the cytostatic agent increases immediately after a single HBO treatment (more than 50%), but not 24 hours after the last repeated HBO treatment. Thus, the uptake is most likely related to the transient increase in oxygenation in the tumor tissue. Factors like tumor P and if collagen content, which decreased significantly in the tumor interstitium after repeated HBO treatment, was without effect on the drug uptake. Conclusion: We showed that hyperoxia increases the uptake of [ H]-5FU in DMBA-induced mammary tumors per se, independently of changes in P , oxygen stress, collagen fibril density, or if transendothelial transport alone. The mechanism by which such an uptake occur is still not elucidated, but it is clearly stimulated by elevated pO . Page 1 of 9 (page number not for citation purposes) BMC Cancer 2009, 9:446 http://www.biomedcentral.com/1471-2407/9/446 ments (each 90 min) of 2 bar pure oxygen on the uptake Background A tumor is comprised of cancer cells as well as stromal of radioactively labelled 5FU in the tumor tissue per se, and micro- cells (fibroblasts, immune cells) that are embedded in an and whether this is related to elevated pO extracellular matrix (ECM) and nourished by vasculature. environmental factors like P , collagen fibrils, transcapil- if Because of irregular and tortuous tumor blood vessels lary transport and oxygen stress. We would then be able to with impaired blood flow and high proliferation rate, conclude that the HBO enhancement of chemotherapeu- tumors have large hypoxic areas, especially in the central tic effects, previously shown in the literature, is not only parts. It is now widely accepted that hypoxia induces due to an enhanced sensitisation of the tumor cells. tumor growth and enhances both radiation- and chemo- resistance of cancer cells [1]. Methods Animals and tumor model Inefficiency of chemotherapy can partly be explained by Female Sprague-Dawley rats were used. Mammary tumors development of multidrug resistance to different chemo- (adenocarcinomas) were induced by dimetyl-α-benzan- therapeutic agents. However, the causes of hypoxia-medi- tracene (DMBA) dissolved in olive oil and given to the rat ated resistance are multifactorial. Some chemotherapeutic by gavage at the age of 7 weeks at a dose of 16 mg (Møl- drugs require oxygen to generate free oxygen radicals that legård, Denmark). The experiments were performed when in turn induce cytotoxicity. Genetic and proteomic the rats were 13-15 weeks old, having reached a body- changes may have substantial effect, by altering prolifera- weight of approximately 250 g and developed one to three tion kinetics, cell cycle position, inhibiting apoptosis and tumors along the mammary crest. Thus, n is equal to regulate angiogenesis and cellular metabolism [2]. How- number of tumors, not the number of rats in these exper- ever, drug resistance can also be caused by the inefficient iments. This was done to minimize the number of ani- transport of the anticancer drug into the tumor tissue. mals used. As tumors are very heterogenous, this was not Solid tumors have a pathologically increased interstitial considered a problem. We did not perform a power anal- fluid pressure (P ) and a dense ECM that make transport ysis prior to the experiments. The number of animals in if of chemotherapeutic agents difficult [3-7]. Increased P each group is determined based on previous experience if leads to decreased transcapillary transport, and thereby with rat experiments. However, this is always a general hinders efficient uptake of chemotherapy [8], while the compromise between experimental accuracy and at the fibrotic nature of the dense ECM in solid tumors have same time keeping the number of animals as low as pos- been shown to impede transport of molecules in the sible. The previous variance in animal experiments has tumor interstitium, and thereby decrease the effect of resulted in the minimum number of animals to n = 5. The cytostatic drugs [9-11]. Since P can be lowered and the animals were randomly allocated to the treatments if structure of the tumor interstitium can be altered, this groups, and none were excluded from the treatment pro- could have the potential to enhance the efficiency of drug- cedure. Malondialdehyde and hydoxyproline experiments based treatment of solid malignancies. were performed blinded. As the main investigator per- formed both the HBO treatment and the rest of the fol- As hypoxia reduces the response of chemotherapeutic low-up experiments, blinding was difficult to accomplish. agents, we aimed to study the effect of enhanced oxygen- All the experiments were performed in accordance with ation on drug-uptake in mammary tumors, by using recommendations of the Norwegian State Commission hyperbaric oxygen (HBO). HBO increases oxygen tension for Laboratory Animals and were approved by the local and oxygen delivery to tissues independent of haemo- ethical committee. globin. This pO elevation has been shown to last for up to 60 min post HBO treatment [12]. Because of this, HBO Hyperbaric hyperbaric chamber has previously been used to enhance the pO in hypoxic A 30 L pressure chamber with an inner diameter of 25 cm, tumor tissue, to potentiate the effect of radio- and photo- and an inner length of 65 cm was used. The chamber was therapy in both clinical and preclinical trials and also the supplied with pure O , and the oxygen concentration (%) effect of some forms of chemotherapy like doxorubicin, was monitored continuously by an oxygen meter (NUI, alkylating agents and 5FU [13-16]. This effect has been Bergen, Norway). After reaching 100% O within approx- ascribed as increased cytotoxicity of the tumor cells to the imately 10 min, the pressure was raised over a period of chemotherapeutic drug in vitro [16,17] and enhanced neo- approximately 3 min to 2 bar. The 2 bar pure oxygen vascularisation in vivo [18]. However, recent studies have atmosphere was maintained for a period of 90 min. To shown reduced vascularisation after HBO treatment in maintain >97% O atmosphere at all times, the chamber vivo [19-21]. was flushed with pure oxygen for 3-5 min every 10-30 min depending on the number of animals in the chamber. The Therefore, the aim of the present study was to study if rats were then decompressed over a period of 10 min. there is an effect of either a single (1) or repeated (4) treat- Page 2 of 9 (page number not for citation purposes) BMC Cancer 2009, 9:446 http://www.biomedcentral.com/1471-2407/9/446 Experimental groups and treatment design The rats were therefore operated before the single 90 min The different experimental groups and their treatment HBO exposure, and left to stabilize for 15 min post expo- H]-5FU was injected into the fem- details are given in Table 1. The repeated HBO treated rats sure. 0.2 ml 0.37 MBq [ were exposed to HBO on day 1, 4, 7 and 10, and the fol- oral catheter, and dialysate was sampled for the next 70 low-up experiments were performed on day 11. The min, analogue to the standard microdialysis experiments. choice of this treatment protocol is due to a previous Measurement of interstitial fluid pressure (P ) study [20], showing that 4 HBO treatments gave maximal if growth-inhibitory effect. The single HBO treated group P was measured using the wick-in-needle (WIN) tech- if was only exposed to HBO on day 1 and measurements nique [7,24]. Briefly, a standard 23-gauge needle, with a 2- performed immediately after the exposure. 4 mm long sidehole, filled with nylon floss and saline was inserted into the central part of the tumor and connected Dose response of 5FU to a transducer dome through a PE-50 catheter. The fluid We tested 3 different doses of 5FU. Original dose (1.5 mg/ communication between the interstitium and the measur- kg), 1/3 (0.5 mg/kg) dose and 3× (4.5 mg/kg) dose 5FU ing system was confirmed by compression and decom- was injected intraperitonally and tumor size was meas- pression of the catheter (clamping). This caused a ured by a calliper on day 1, 4, 7 and 11, and estimated transient rise and fall in the pressure. A measurement was according to the formula: V = π/6 (a ·b), where 'a' accepted when the pressure returned to pre-clamp value denotes the shortest transversal diameter and 'b' the long- (± 1 mmHg). est transversal diameter. Growth was calculated as growth change compared to day 1 (100%). Determination of collagen content in the tumors Hydroxyproline (to estimate collagen content) was deter- Microdialysis mined in acid hydrolysates of the tumor tissue by a colo- To determine the uptake of radioactively labelled 5FU metric method adapted from Woessner et al. [25]. ([ H]-5FU) (Nycomed Amersham, Buckinghamshire, UK) into the tumor tissue, microdialysis technique [22] was Oxygen stress measured by Malondialdehyd (MDA) used, as modified in our laboratory [23]. After anaesthe- To evaluate oxidative stress on lipids and membranes (the H]- status of oxidative damage) we measured the lipid perox- sia, the femoral vein was cannulated for injection of [ 5FU. One microdialysis probe (CMA/20, Microdialysis idation product MDA, in controls and HBO treated AB, Stockholm, Sweden) was placed in the jugular vein tumors. MDA was analysed by a spectrophotometric assay and one in the tumor. Both probes were connected to a according to the manufacturer's protocol (Northwest Life pump (CMA-100, Microdialysis AB, Stockholm, Sweden) Science Specialties, LCC, Vancouver). The level of thiobar- and the catheters were perfused with saline at a rate of 1 bituric reactive substances (TBARS) was assessed in tissue μl/min. The catheter and probes were left to stabilize and homogenates (10% w/v prepared from frozen samples). rd equilibrate for 60 min before sampling of dialysate. Sam- As recommended for complex samples, the 3 -derivative pling of dialysate from both tumor and plasma started spectra (400-700 nm) were obtained, and MDA levels immediately after injection of 0.2 ml 0.37 Mbq [ H]-5FU were calculated based on the peak value around 510-520 and fractions were collected every 10 min for a total of 70 nm min. The area under the curve (AUC) for the plasma and Volume calculations by the dilution principle tumor was calculated as the product of counts per 10 min (cpm) for a total measurement period of 70 min. Trans- Extracellular volume (ECV) and plasma volume (PV) in port of [ H]-5FU was expressed as AUC tumor divided by tumor and skin was measured by radioactively labelled AUC plasma. isotopes using the dilution principle as previously described [26]. Total tissue water (TTW) was calculated as: An additional, single HBO exposure group was performed (wet weight-dry weight)/(dry weight). Samples were dried to elucidate if the generally known enhanced effect of at 50°C until constant weight. The distribution volumes cytostatic agents in combination with HBO is due to the for Cr-EDTA (Institute of Energy Technology, Kjeller, 125 125 high pO during and immediately after HBO exposure. Norway) and I-labeled human serum albumin ( I- Table 1: The experimental groups. Experimental groups Gas Ambient pressure pO Number of exposures Exposure time Control air 1 bar 0.2 - - Repeated HBO treatment O 2 bar 2.0 4 90 min Single HBO treatment O 2 bar 2.0 1 90 min Page 3 of 9 (page number not for citation purposes) BMC Cancer 2009, 9:446 http://www.biomedcentral.com/1471-2407/9/446 HSA) (Institute of Energy Technology, Kjeller, Norway), into the tumor tissue 24 hours after the last repeated HBO measuring ECV and PV respectively, were calculated as treatment (n = 8) compared to control (n = 7). However, immediately after the single HBO treatment (n = 5) there plasma equivalent volumes, i.e. counts per min per mg of tissue divided by counts per min per ml of plasma. Both was a significant increase in uptake (p < 0.05) (Table 2). isotopes were given after functional nephrectomy by bilat- eral ligation of the renal pedicles via flank incision. Cr- Hyperoxia lowers interstitial fluid pressure (P ) if EDTA (0.7 MBq) was injected into the jugular catheter As elevated P has been proposed to inhibit the transport if and left to circulate for 85 min before injecting 0.05 MBq and effect of chemotherapy, we wanted to elucidate if this I-HSA. Blood samples were taken 5 min later, by heart was the case also for DMBA induced mammary tumors. puncture. The P is usually high in solid tumors, reaching 5.5 ± 0.9 if mmHg in the control tumors (n = 9) (Table 2). However, Immunohistochemistry the average P after both single (n = 17) and repeated if The animals were sacrificed with saturated KCl during HBO (n = 13) treatment was significantly reduced (p < anaesthesia and the tumors were immediately dissected 0.05) compared to control. and then stored in -80°C. out and put into liquid N Hyperoxia influences collagen fibrils Frozen tumor sections (20 μm) were used for studying A dense collagen network has been proposed to hinder lymph-vessel morphology. The sections were stained with efficient transport of chemotherapeutic drugs. Therefore an antibody against lymphatic vessel endothelial hyaluro- we have elucidated collagen content in both control and nan receptor-1 (LYVE-1) with the Avidin-Biotin Complex HBO treated tumors. The amount of hydroxyproline was (ABC) method, using a commercially available kit (Vector significantly lower in the HBO treated tumors compared Vectastatin Elite ABC Staining Kit, Vector Laboratories to controls, indicating a corresponding decrease in colla- Inc). Diaminobenzidine (DAB) was used as a chromogen gen content (Table 2). to visualize the lymph-vessels. Finally, the sections were counterstained with Richardsons dye. The sections were Hyperoxia and oxygen radicals examined using a Nikon light microscope (THP Eclipse As hyperoxia might induce elevated oxygen radical levels, it was desirable to measure if this could have influenced E600, Nikon Corporation, Tokyo, Japan) and the images were captured with a Nikon Digital Camera (DXM 1200F, the effect of chemotherapy after HBO treatment. Measure- Nikon Corporation, Tokyo, Japan). ments of MDA-levels showed no significant differences in oxygen radicals compared to control (n = 5), neither in Statistics the acute (n = 5) nor the repeated (n = 5) HBO treated All data were tested for normality prior to the choice of group (Table 2). The higher mean value in the repeated statistical analysis. Results were analysed statistically group is due to one diverging value. Discarding this value using two-way analysis with unpaired t-test comparing would give an identical mean as to the two other groups. results from the groups. Paired t-test was used when com- paring results within the same group of animals. Non-par- Extracellular fluid volume, plasma volume and total tissue ametric Mann-Whitney test was used for the P water if measurements, while ANOVA with Tukey post test was Since P in the HBO treated tumors was lowered, we if used to compare the results from the microdialysis. A assumed that this could influence the fluid distribution. value of p < 0.05 was considered statistically significant. However, there was no statistically significant difference in ECV, PV or TTW in either the tumor or skin tissue Results between control (n = 9) and repeated HBO treated rats (n Dose response of 5FU = 8) (Table 3). To examine if different doses of the chemotherapeutic drug might influence the effect on tumor growth, dose Hyperoxia influenced tumor lymphatics response of 3 different doses were tested. However, there As the amount and functionality of lymph vessels influ- was no significant difference in tumor growth inhibition ence both P , fluid distribution and possibly the transport if between the three different doses of 5FU (Figure 1). (n = of chemotherapy, we stained lymph vessels to elucidate 5 for each group). possible changes after HBO. Staining with LYVE-1 showed continuous lymphatic vessels in the mammary control Uptake of [ H]-5FU tumors, located in the connective tissue close to larger Microdialysis was used to examine if oxygenation of the blood vessels. After HBO treatment, however, the lym- tumor after HBO treatment influenced the direct uptake phatic vessels are disintegrated and no longer continuous of the chemotherapeutic drug into the tumor tissue. (Figure 2). Microdialysis showed no increase in uptake of [ H]-5FU Page 4 of 9 (page number not for citation purposes) BMC Cancer 2009, 9:446 http://www.biomedcentral.com/1471-2407/9/446 Th 3 Figure 1 × (4.5 e effect of mg/kgth ) dose ree different doses of 5-fluorouracil (5FU) on tumor growth, original dose (1.5 mg/kg), 1/3 (0.5 mg/kg) dose and The effect of three different doses of 5-fluorouracil (5FU) on tumor growth, original dose (1.5 mg/kg), 1/3 (0.5 mg/kg) dose and 3× (4.5 mg/kg) dose. Tumor growth is measured as % of initial volume. Mean ± SD. interstitium, further transport is heavily influenced by the Discussion Stuhr et al. [14] demonstrated increased effect of the com- organization and conductivity, and thereby the composi- monly used cytostatic drug 5FU in DMBA-induced mam- tion of the interstitium. Several investigators have shown mary tumors, when injected intraperitoneally increased uptake of cytostatic agents after decreased immediately before each HBO treatment. Dose-response tumor P [8,29-32]. In a recent review, Heldin et al. [8] if experiments in this study showed no statistical difference suggest that increased P contributes to a decreased capil- if lary transport in tumors, to hinder efficient uptake of between 1/3×, 1× and 3× dose of 5FU (Figure 1), indicat- ing that it must be the HBO treatment that potentiated the drugs into the tumor tissue. Additionally, the composi- effect of the chemotherapeutic drug. We therefore wanted tion and organisation of the ECM, cell-cell interactions, to investigate if there is an effect of either a single or and the tumor structure also affect drug penetration [33]. repeated treatment of 2 bar pure oxygen on the uptake of Eikenes et al. [11] proposed that the structure of the ECM radioactively labelled 5FU in the tumor tissue per se. We is the single most important factor for macromolecule have concluded that the potentiation of the chemothera- uptake. In accordance with this, Netti et al. [9], found an peutic effect after HBO is not only due to an enhanced unexpected correspondence between transport resistance cytotoxicity of the tumor cells, as has been previously pos- and the mechanical stiffness of the ECM, with an tulated in the literature [15,27,28], but also due to an extended collagen network in the more penetration-resist- direct increase in uptake of the chemotherapeutic drug. ant tumors. Also, others have shown that tumors with high collagen density and small interstitial space have Successful delivery of systemically administered cytostatic lower drug penetration [34,35]. Furthermore, increased agents to the tumor tissue requires that the drug must blood flow and blood vessel permeability are factors that reach the target cells in optimal dosage and in addition be are important in drug-uptake [36]. Thus, we chose espe- effective in the tumor microenvironment. After a mole- cially to investigate the relation between uptake of the cule has been moved out of the blood vessels and into the Page 5 of 9 (page number not for citation purposes) BMC Cancer 2009, 9:446 http://www.biomedcentral.com/1471-2407/9/446 Table 2: Uptake of [ H]-5-fluorouracil (5FU), interstitial fluid pressure (P ), collagen content and malondialdehyd (MDA) levels in if controls and after repeated or single hyperbaric oxygen exposure (HBO). Control Repeated HBO Single HBO Uptake of [ H]-5FU 0.17 ± 0.12 0.20 ± 0.11 0.42 ± 0.21 * (AUC tumor/AUC plasma) (n = 7) (n = 8) (n = 5) P 5.5 ± 2.6 2.3 ± 2.7* 2.5 ± 2.9* if (mmHg) (n = 9) (n = 11) (n = 17) %collagen of dry weight 11.6 ± 5.0 5.0 ± 1.1* (n = 6) (n = 4) MDA levels 0.09 ± 0.01 0.15 ± 0.11 0.09 ± 0.02 (nmol/mg prot) (n = 5) (n = 5) (n = 5) Mean ± SD. * p < 0.05 vs control cytostatic drug, transendothelial transport, tumor P and fibrils is reduced, as shown with hydroxyproline quantifi- if the collagen fibril network. cation, this effect would not be as pronounced, and the P if might be reduced. Thus, it seems like repeated HBO treat- We found a significant reduction in tumor P after both ment have reduced tumor P by reducing collagen content if if acute and repeated HBO treatment, and expected that this and density, most likely by enhancing breakdown of col- would be reflected in an increased uptake of [ H]-5FU lagen. after both treatments. This is, however, not the fact in our study, where the uptake of [ H]-5FU was significantly Tumor vessels are hyperpermeable due to up-regulation of increased after single HBO treatment, but not after the vascular mediators such as nitric oxide, bradykinin as well repeated HBO treatment. The cpm-ratio between tumor as anatomic defects like large gap junctions between adja- and plasma was stable over the 70 min sampling-period cent cells and lack of pericytes [36,39]. An explanation for H]-5FU in the repeated HBO during microdialysis for the single HBO treatment, indi- the lack of uptake of [ cating a constant condition determining the uptake. The treated tumors, might be the proposed mechanism by Lee increased uptake of the drug into the tumor tissue, after et al. [40] of "normalization" of the abnormal structure single HBO exposure is most likely influenced by and function of the tumor vasculature after anti-ang- increased pO . Previous studies reported enhanced pO iogenic treatment. We have previously shown that 2 2 for up to one hour after HBO treatment [12,37]. Since the repeated HBO induces an anti-angiogenic effect in the repeated HBO treated animals were measured 24 hours DMBA induced tumors [19,21]. Lee et al. [40] suggested after the last HBO treatment, the pO is normalized and that it is the quality of the vascular organization and not any effect of pO will not be measurable. In the single just the quantity of the vessels that determines the vessel HBO experiments, however, the pO is still high. The function, and that the loss of endothelial cells would repeated HBO treatment probably induces more long- reduce the tortuousity of vessels or eliminate them alto- term changes in the tumor tissue that influences P . The gether. Later, several preclinical and clinical studies if reduction in collagen may be involved in the reduction of [41,42] have shown that the "normalized" vasculature P over time. Stromal fibroblasts are able to exert tension after anti-angiogenic therapy had less leaky and tortuous if on the collagen microfibrillar network through the colla- vessels, with more normal basement membranes and bet- gen binding integrins [38]. When the amount of collagen ter pericyte coverage, and that these structural changes Table 3: The extracellular volume (ECV), plasma volume (PV) and total tissue water (TTW) in skin and tumor in control and after repeated hyperbaric oxygen (HBO) exposure. ECV PV TTW (ml/g dry weight) (ml/g dry weight) (ml/g dry weight) Skin Tumor Skin Tumor Skin Tumor Control 0.55 ± 0.12 1.00 ± 0.27 0.007 ± 0.004 0.07 ± 0.04 1.44 ± 0.09 3.73 ± 0.45 (n = 9) HBO 0.50 ± 0.18 0.85 ± 0.47 0.004 ± 0.003 0.06 ± 0.03 1.45 ± 0.2 4.05 ± 0.9 (n = 8) Means ± SD. Page 6 of 9 (page number not for citation purposes) BMC Cancer 2009, 9:446 http://www.biomedcentral.com/1471-2407/9/446 Lymph vessels an Figure 2 d D) stained with LYVE-1 in two different control tumors (A and C) and hyperbaric oxygen (HBO) treated tumors (B Lymph vessels stained with LYVE-1 in two different control tumors (A and C) and hyperbaric oxygen (HBO) treated tumors (B and D). The staining was performed concomitant in the two groups. Note disintegration of lymphatics after HBO treatment, and seemingly anatomically normal lymphatics in untreated control tumors. were accompanied by "normalization" of the tumor In normal tissue, the interstitial fluid volume is kept fairly microenvironment. This modification in vascular archi- constant by several mechanisms [46], such as lymph flow tecture would decrease vascular permeability and reduce and adjustment of pressures acting across the capillary flow resistance and hence lower both mean venous pres- wall [47]. LYVE-1 surprisingly shows continuous lymph sure and P [8]. Experimental studies have demonstrated vessels in our control tumors, but dissolved lymph vessels if that anti-angiogenic therapy can decrease the overall dis- after repeated HBO treatment. However, Fukumura et al. tribution of large macromolecules such as antibodies for [48] stated that even though the structures with lymphatic instance [43,44] and decrease blood perfusion [45]. Thus, endothelial marker are present in tumors, they probably since we have previously shown that HBO has an anti- do not transport fluid or macromolecules. This, together angiogenic effect on DMBA induced tumors after repeated with the observed lowering of tumor P after repeated if HBO treatment [19], this will together with a possible HBO treatment, should lead to an increase in ECV. Never- reduced capillary permeability, impede transendothelial theless, ECV, PV and TTW did not change in tumors after transport of [ H]-5FU, even though P is lowered. repeated HBO treatment compared to control, indicating if that neither the lowered P nor the disintegrated lymphat- if ics after HBO contribute to the unchanged ECV found in Page 7 of 9 (page number not for citation purposes) BMC Cancer 2009, 9:446 http://www.biomedcentral.com/1471-2407/9/446 the present study. As already mentioned, the possible ume experiments. RKR and LS participated in the study "normalization" of the tumor vasculature, after repeated design, interpretation of data and manuscript drafting. All HBO treatment, is expected to normalize capillary perme- authors read and approved the final manuscript. ability, and thereby prevent any increase in ECV, even though P is decreased. Acknowledgements if This study was supported by grants from Helse Vest (Grants 911370), Edel and Ole Stakvold's foundation and The Norwegian cancer society. Ingrid Reactive oxygen species and oxidative stress are consid- Strand and Åse Rye Eriksen are gratefully acknowledged for technical assist- ered to be important in several aspects of malignancies, ance. both in tumor development as well as therapeutic strate- gies such as radiotherapy [49]. Since changes in oxygen References concentration may affect the production of its reactive 1. Hoogsteen IJ, Marres HA, Kogel AJ van der, Kaanders JH: The derivatives in the tumor, it is relevant to suggest that anti- hypoxic tumour microenvironment, patient selection and hypoxia-modifying treatments. Clin Oncol (R Coll Radiol) 2007, tumor effects of HBO may be due to oxidative stress. Fur- 19(6):385-396. thermore, as stated in the introduction, some 2. 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Nat Rev Cancer 2004, 4(11):891-899. Sjoquist M, Ahlstrom H, Reed RK, Rubin K, Lammerts E, Roswall P, Gotwals PJ, Koteliansky VE, Heldin NE: Lowering of tumor inter- Pre-publication history stitial fluid pressure specifically augments efficacy of chemo- therapy. Interference with TGF-beta1 and -beta3 in tumor The pre-publication history for this paper can be accessed stroma lowers tumor interstitial fluid pressure independ- here: ently of growth in experimental carcinoma. Faseb J 2003, 17(12):1756-1758. 32. Stuhr LEB, Salnikov AV, Iversen VV, Salvesen G, Rubin K, Reed RK: http://www.biomedcentral.com/1471-2407/9/446/pre High-dose, short-term, anti-inflammatory treatment with pub dexamethasone reduces growth and augments the effects of 5-fluorouracil on dimethyl-alpha-benzanthracene-induced mammary tumors in rats. Scand J Clin Lab Invest 2006, 66:477-486. 33. Davies E, Clarke C, Hopkins A: Malignant cerebral glioma-I: Sur- vival, disability, and morbidity after radiotherapy. BMJ 1996, 313:1507-1512. 34. Kuh HJ, Jang SH, Wientjes MG, Weaver JR, Au JL: Determinants of paclitaxel penetration and accumulation in human solid tumor. J Pharmacol Exp Ther 1999, 290(2):871-880. 35. Au JL, Jang SH, Wientjes MG: Clinical aspects of drug delivery to tumors. J Control Release 2002, 78(1-3):81-95. 36. Jain RK: Delivery of molecular and cellular medicine to solid tumors. J Cont Release 1998, 53(1-3):49-67. 37. Brizel DM, Lin S, Johnson JL, Brooks J, Dewhirst MW, Piantadosi CA: The mechanisms by which hyperbaric oxygen and carbogen Page 9 of 9 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Cancer Springer Journals

Hyperoxia increases the uptake of 5-fluorouracil in mammary tumors independently of changes in interstitial fluid pressure and tumor stroma

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Springer Journals
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Copyright © 2009 by Moen et al; licensee BioMed Central Ltd.
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Biomedicine; Cancer Research; Oncology; Surgical Oncology; Health Promotion and Disease Prevention; Biomedicine general; Medicine/Public Health, general
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1471-2407
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1471-2407
DOI
10.1186/1471-2407-9-446
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20017908
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Abstract

Background: Hypoxia is associated with increased resistance to chemo- and radiation-therapy. Hyperoxic treatment (hyperbaric oxygen) has previously been shown to potentiate the effect of some forms of chemotherapy, and this has been ascribed to enhanced cytotoxicity or neovascularisation. The aim of this study was to elucidate whether hyperoxia also enhances any actual uptake of 5FU (5-fluorouracil) into the tumor tissue and if this can be explained by changes in the interstitium and extracellular matrix. Methods: One group of tumor bearing rats was exposed to repeated hyperbaric oxygen (HBO) treatment (2 bar, pO = 2 bar, 4 exposures à 90 min), whereas one group was exposed to one single identical HBO treatment. Animals housed under normal atmosphere (1 bar, pO = 0.2 bar) served as controls. Three doses of 5FU were tested for dose response. Uptake of [ H]-5FU in the tumor was assessed, with special reference to factors that might have contributed, such as interstitial fluid pressure (P ), collagen content, oxygen stress (measured as malondialdehyd levels), if lymphatics and transcapillary transport in the tumors. Results: The uptake of the cytostatic agent increases immediately after a single HBO treatment (more than 50%), but not 24 hours after the last repeated HBO treatment. Thus, the uptake is most likely related to the transient increase in oxygenation in the tumor tissue. Factors like tumor P and if collagen content, which decreased significantly in the tumor interstitium after repeated HBO treatment, was without effect on the drug uptake. Conclusion: We showed that hyperoxia increases the uptake of [ H]-5FU in DMBA-induced mammary tumors per se, independently of changes in P , oxygen stress, collagen fibril density, or if transendothelial transport alone. The mechanism by which such an uptake occur is still not elucidated, but it is clearly stimulated by elevated pO . Page 1 of 9 (page number not for citation purposes) BMC Cancer 2009, 9:446 http://www.biomedcentral.com/1471-2407/9/446 ments (each 90 min) of 2 bar pure oxygen on the uptake Background A tumor is comprised of cancer cells as well as stromal of radioactively labelled 5FU in the tumor tissue per se, and micro- cells (fibroblasts, immune cells) that are embedded in an and whether this is related to elevated pO extracellular matrix (ECM) and nourished by vasculature. environmental factors like P , collagen fibrils, transcapil- if Because of irregular and tortuous tumor blood vessels lary transport and oxygen stress. We would then be able to with impaired blood flow and high proliferation rate, conclude that the HBO enhancement of chemotherapeu- tumors have large hypoxic areas, especially in the central tic effects, previously shown in the literature, is not only parts. It is now widely accepted that hypoxia induces due to an enhanced sensitisation of the tumor cells. tumor growth and enhances both radiation- and chemo- resistance of cancer cells [1]. Methods Animals and tumor model Inefficiency of chemotherapy can partly be explained by Female Sprague-Dawley rats were used. Mammary tumors development of multidrug resistance to different chemo- (adenocarcinomas) were induced by dimetyl-α-benzan- therapeutic agents. However, the causes of hypoxia-medi- tracene (DMBA) dissolved in olive oil and given to the rat ated resistance are multifactorial. Some chemotherapeutic by gavage at the age of 7 weeks at a dose of 16 mg (Møl- drugs require oxygen to generate free oxygen radicals that legård, Denmark). The experiments were performed when in turn induce cytotoxicity. Genetic and proteomic the rats were 13-15 weeks old, having reached a body- changes may have substantial effect, by altering prolifera- weight of approximately 250 g and developed one to three tion kinetics, cell cycle position, inhibiting apoptosis and tumors along the mammary crest. Thus, n is equal to regulate angiogenesis and cellular metabolism [2]. How- number of tumors, not the number of rats in these exper- ever, drug resistance can also be caused by the inefficient iments. This was done to minimize the number of ani- transport of the anticancer drug into the tumor tissue. mals used. As tumors are very heterogenous, this was not Solid tumors have a pathologically increased interstitial considered a problem. We did not perform a power anal- fluid pressure (P ) and a dense ECM that make transport ysis prior to the experiments. The number of animals in if of chemotherapeutic agents difficult [3-7]. Increased P each group is determined based on previous experience if leads to decreased transcapillary transport, and thereby with rat experiments. However, this is always a general hinders efficient uptake of chemotherapy [8], while the compromise between experimental accuracy and at the fibrotic nature of the dense ECM in solid tumors have same time keeping the number of animals as low as pos- been shown to impede transport of molecules in the sible. The previous variance in animal experiments has tumor interstitium, and thereby decrease the effect of resulted in the minimum number of animals to n = 5. The cytostatic drugs [9-11]. Since P can be lowered and the animals were randomly allocated to the treatments if structure of the tumor interstitium can be altered, this groups, and none were excluded from the treatment pro- could have the potential to enhance the efficiency of drug- cedure. Malondialdehyde and hydoxyproline experiments based treatment of solid malignancies. were performed blinded. As the main investigator per- formed both the HBO treatment and the rest of the fol- As hypoxia reduces the response of chemotherapeutic low-up experiments, blinding was difficult to accomplish. agents, we aimed to study the effect of enhanced oxygen- All the experiments were performed in accordance with ation on drug-uptake in mammary tumors, by using recommendations of the Norwegian State Commission hyperbaric oxygen (HBO). HBO increases oxygen tension for Laboratory Animals and were approved by the local and oxygen delivery to tissues independent of haemo- ethical committee. globin. This pO elevation has been shown to last for up to 60 min post HBO treatment [12]. Because of this, HBO Hyperbaric hyperbaric chamber has previously been used to enhance the pO in hypoxic A 30 L pressure chamber with an inner diameter of 25 cm, tumor tissue, to potentiate the effect of radio- and photo- and an inner length of 65 cm was used. The chamber was therapy in both clinical and preclinical trials and also the supplied with pure O , and the oxygen concentration (%) effect of some forms of chemotherapy like doxorubicin, was monitored continuously by an oxygen meter (NUI, alkylating agents and 5FU [13-16]. This effect has been Bergen, Norway). After reaching 100% O within approx- ascribed as increased cytotoxicity of the tumor cells to the imately 10 min, the pressure was raised over a period of chemotherapeutic drug in vitro [16,17] and enhanced neo- approximately 3 min to 2 bar. The 2 bar pure oxygen vascularisation in vivo [18]. However, recent studies have atmosphere was maintained for a period of 90 min. To shown reduced vascularisation after HBO treatment in maintain >97% O atmosphere at all times, the chamber vivo [19-21]. was flushed with pure oxygen for 3-5 min every 10-30 min depending on the number of animals in the chamber. The Therefore, the aim of the present study was to study if rats were then decompressed over a period of 10 min. there is an effect of either a single (1) or repeated (4) treat- Page 2 of 9 (page number not for citation purposes) BMC Cancer 2009, 9:446 http://www.biomedcentral.com/1471-2407/9/446 Experimental groups and treatment design The rats were therefore operated before the single 90 min The different experimental groups and their treatment HBO exposure, and left to stabilize for 15 min post expo- H]-5FU was injected into the fem- details are given in Table 1. The repeated HBO treated rats sure. 0.2 ml 0.37 MBq [ were exposed to HBO on day 1, 4, 7 and 10, and the fol- oral catheter, and dialysate was sampled for the next 70 low-up experiments were performed on day 11. The min, analogue to the standard microdialysis experiments. choice of this treatment protocol is due to a previous Measurement of interstitial fluid pressure (P ) study [20], showing that 4 HBO treatments gave maximal if growth-inhibitory effect. The single HBO treated group P was measured using the wick-in-needle (WIN) tech- if was only exposed to HBO on day 1 and measurements nique [7,24]. Briefly, a standard 23-gauge needle, with a 2- performed immediately after the exposure. 4 mm long sidehole, filled with nylon floss and saline was inserted into the central part of the tumor and connected Dose response of 5FU to a transducer dome through a PE-50 catheter. The fluid We tested 3 different doses of 5FU. Original dose (1.5 mg/ communication between the interstitium and the measur- kg), 1/3 (0.5 mg/kg) dose and 3× (4.5 mg/kg) dose 5FU ing system was confirmed by compression and decom- was injected intraperitonally and tumor size was meas- pression of the catheter (clamping). This caused a ured by a calliper on day 1, 4, 7 and 11, and estimated transient rise and fall in the pressure. A measurement was according to the formula: V = π/6 (a ·b), where 'a' accepted when the pressure returned to pre-clamp value denotes the shortest transversal diameter and 'b' the long- (± 1 mmHg). est transversal diameter. Growth was calculated as growth change compared to day 1 (100%). Determination of collagen content in the tumors Hydroxyproline (to estimate collagen content) was deter- Microdialysis mined in acid hydrolysates of the tumor tissue by a colo- To determine the uptake of radioactively labelled 5FU metric method adapted from Woessner et al. [25]. ([ H]-5FU) (Nycomed Amersham, Buckinghamshire, UK) into the tumor tissue, microdialysis technique [22] was Oxygen stress measured by Malondialdehyd (MDA) used, as modified in our laboratory [23]. After anaesthe- To evaluate oxidative stress on lipids and membranes (the H]- status of oxidative damage) we measured the lipid perox- sia, the femoral vein was cannulated for injection of [ 5FU. One microdialysis probe (CMA/20, Microdialysis idation product MDA, in controls and HBO treated AB, Stockholm, Sweden) was placed in the jugular vein tumors. MDA was analysed by a spectrophotometric assay and one in the tumor. Both probes were connected to a according to the manufacturer's protocol (Northwest Life pump (CMA-100, Microdialysis AB, Stockholm, Sweden) Science Specialties, LCC, Vancouver). The level of thiobar- and the catheters were perfused with saline at a rate of 1 bituric reactive substances (TBARS) was assessed in tissue μl/min. The catheter and probes were left to stabilize and homogenates (10% w/v prepared from frozen samples). rd equilibrate for 60 min before sampling of dialysate. Sam- As recommended for complex samples, the 3 -derivative pling of dialysate from both tumor and plasma started spectra (400-700 nm) were obtained, and MDA levels immediately after injection of 0.2 ml 0.37 Mbq [ H]-5FU were calculated based on the peak value around 510-520 and fractions were collected every 10 min for a total of 70 nm min. The area under the curve (AUC) for the plasma and Volume calculations by the dilution principle tumor was calculated as the product of counts per 10 min (cpm) for a total measurement period of 70 min. Trans- Extracellular volume (ECV) and plasma volume (PV) in port of [ H]-5FU was expressed as AUC tumor divided by tumor and skin was measured by radioactively labelled AUC plasma. isotopes using the dilution principle as previously described [26]. Total tissue water (TTW) was calculated as: An additional, single HBO exposure group was performed (wet weight-dry weight)/(dry weight). Samples were dried to elucidate if the generally known enhanced effect of at 50°C until constant weight. The distribution volumes cytostatic agents in combination with HBO is due to the for Cr-EDTA (Institute of Energy Technology, Kjeller, 125 125 high pO during and immediately after HBO exposure. Norway) and I-labeled human serum albumin ( I- Table 1: The experimental groups. Experimental groups Gas Ambient pressure pO Number of exposures Exposure time Control air 1 bar 0.2 - - Repeated HBO treatment O 2 bar 2.0 4 90 min Single HBO treatment O 2 bar 2.0 1 90 min Page 3 of 9 (page number not for citation purposes) BMC Cancer 2009, 9:446 http://www.biomedcentral.com/1471-2407/9/446 HSA) (Institute of Energy Technology, Kjeller, Norway), into the tumor tissue 24 hours after the last repeated HBO measuring ECV and PV respectively, were calculated as treatment (n = 8) compared to control (n = 7). However, immediately after the single HBO treatment (n = 5) there plasma equivalent volumes, i.e. counts per min per mg of tissue divided by counts per min per ml of plasma. Both was a significant increase in uptake (p < 0.05) (Table 2). isotopes were given after functional nephrectomy by bilat- eral ligation of the renal pedicles via flank incision. Cr- Hyperoxia lowers interstitial fluid pressure (P ) if EDTA (0.7 MBq) was injected into the jugular catheter As elevated P has been proposed to inhibit the transport if and left to circulate for 85 min before injecting 0.05 MBq and effect of chemotherapy, we wanted to elucidate if this I-HSA. Blood samples were taken 5 min later, by heart was the case also for DMBA induced mammary tumors. puncture. The P is usually high in solid tumors, reaching 5.5 ± 0.9 if mmHg in the control tumors (n = 9) (Table 2). However, Immunohistochemistry the average P after both single (n = 17) and repeated if The animals were sacrificed with saturated KCl during HBO (n = 13) treatment was significantly reduced (p < anaesthesia and the tumors were immediately dissected 0.05) compared to control. and then stored in -80°C. out and put into liquid N Hyperoxia influences collagen fibrils Frozen tumor sections (20 μm) were used for studying A dense collagen network has been proposed to hinder lymph-vessel morphology. The sections were stained with efficient transport of chemotherapeutic drugs. Therefore an antibody against lymphatic vessel endothelial hyaluro- we have elucidated collagen content in both control and nan receptor-1 (LYVE-1) with the Avidin-Biotin Complex HBO treated tumors. The amount of hydroxyproline was (ABC) method, using a commercially available kit (Vector significantly lower in the HBO treated tumors compared Vectastatin Elite ABC Staining Kit, Vector Laboratories to controls, indicating a corresponding decrease in colla- Inc). Diaminobenzidine (DAB) was used as a chromogen gen content (Table 2). to visualize the lymph-vessels. Finally, the sections were counterstained with Richardsons dye. The sections were Hyperoxia and oxygen radicals examined using a Nikon light microscope (THP Eclipse As hyperoxia might induce elevated oxygen radical levels, it was desirable to measure if this could have influenced E600, Nikon Corporation, Tokyo, Japan) and the images were captured with a Nikon Digital Camera (DXM 1200F, the effect of chemotherapy after HBO treatment. Measure- Nikon Corporation, Tokyo, Japan). ments of MDA-levels showed no significant differences in oxygen radicals compared to control (n = 5), neither in Statistics the acute (n = 5) nor the repeated (n = 5) HBO treated All data were tested for normality prior to the choice of group (Table 2). The higher mean value in the repeated statistical analysis. Results were analysed statistically group is due to one diverging value. Discarding this value using two-way analysis with unpaired t-test comparing would give an identical mean as to the two other groups. results from the groups. Paired t-test was used when com- paring results within the same group of animals. Non-par- Extracellular fluid volume, plasma volume and total tissue ametric Mann-Whitney test was used for the P water if measurements, while ANOVA with Tukey post test was Since P in the HBO treated tumors was lowered, we if used to compare the results from the microdialysis. A assumed that this could influence the fluid distribution. value of p < 0.05 was considered statistically significant. However, there was no statistically significant difference in ECV, PV or TTW in either the tumor or skin tissue Results between control (n = 9) and repeated HBO treated rats (n Dose response of 5FU = 8) (Table 3). To examine if different doses of the chemotherapeutic drug might influence the effect on tumor growth, dose Hyperoxia influenced tumor lymphatics response of 3 different doses were tested. However, there As the amount and functionality of lymph vessels influ- was no significant difference in tumor growth inhibition ence both P , fluid distribution and possibly the transport if between the three different doses of 5FU (Figure 1). (n = of chemotherapy, we stained lymph vessels to elucidate 5 for each group). possible changes after HBO. Staining with LYVE-1 showed continuous lymphatic vessels in the mammary control Uptake of [ H]-5FU tumors, located in the connective tissue close to larger Microdialysis was used to examine if oxygenation of the blood vessels. After HBO treatment, however, the lym- tumor after HBO treatment influenced the direct uptake phatic vessels are disintegrated and no longer continuous of the chemotherapeutic drug into the tumor tissue. (Figure 2). Microdialysis showed no increase in uptake of [ H]-5FU Page 4 of 9 (page number not for citation purposes) BMC Cancer 2009, 9:446 http://www.biomedcentral.com/1471-2407/9/446 Th 3 Figure 1 × (4.5 e effect of mg/kgth ) dose ree different doses of 5-fluorouracil (5FU) on tumor growth, original dose (1.5 mg/kg), 1/3 (0.5 mg/kg) dose and The effect of three different doses of 5-fluorouracil (5FU) on tumor growth, original dose (1.5 mg/kg), 1/3 (0.5 mg/kg) dose and 3× (4.5 mg/kg) dose. Tumor growth is measured as % of initial volume. Mean ± SD. interstitium, further transport is heavily influenced by the Discussion Stuhr et al. [14] demonstrated increased effect of the com- organization and conductivity, and thereby the composi- monly used cytostatic drug 5FU in DMBA-induced mam- tion of the interstitium. Several investigators have shown mary tumors, when injected intraperitoneally increased uptake of cytostatic agents after decreased immediately before each HBO treatment. Dose-response tumor P [8,29-32]. In a recent review, Heldin et al. [8] if experiments in this study showed no statistical difference suggest that increased P contributes to a decreased capil- if lary transport in tumors, to hinder efficient uptake of between 1/3×, 1× and 3× dose of 5FU (Figure 1), indicat- ing that it must be the HBO treatment that potentiated the drugs into the tumor tissue. Additionally, the composi- effect of the chemotherapeutic drug. We therefore wanted tion and organisation of the ECM, cell-cell interactions, to investigate if there is an effect of either a single or and the tumor structure also affect drug penetration [33]. repeated treatment of 2 bar pure oxygen on the uptake of Eikenes et al. [11] proposed that the structure of the ECM radioactively labelled 5FU in the tumor tissue per se. We is the single most important factor for macromolecule have concluded that the potentiation of the chemothera- uptake. In accordance with this, Netti et al. [9], found an peutic effect after HBO is not only due to an enhanced unexpected correspondence between transport resistance cytotoxicity of the tumor cells, as has been previously pos- and the mechanical stiffness of the ECM, with an tulated in the literature [15,27,28], but also due to an extended collagen network in the more penetration-resist- direct increase in uptake of the chemotherapeutic drug. ant tumors. Also, others have shown that tumors with high collagen density and small interstitial space have Successful delivery of systemically administered cytostatic lower drug penetration [34,35]. Furthermore, increased agents to the tumor tissue requires that the drug must blood flow and blood vessel permeability are factors that reach the target cells in optimal dosage and in addition be are important in drug-uptake [36]. Thus, we chose espe- effective in the tumor microenvironment. After a mole- cially to investigate the relation between uptake of the cule has been moved out of the blood vessels and into the Page 5 of 9 (page number not for citation purposes) BMC Cancer 2009, 9:446 http://www.biomedcentral.com/1471-2407/9/446 Table 2: Uptake of [ H]-5-fluorouracil (5FU), interstitial fluid pressure (P ), collagen content and malondialdehyd (MDA) levels in if controls and after repeated or single hyperbaric oxygen exposure (HBO). Control Repeated HBO Single HBO Uptake of [ H]-5FU 0.17 ± 0.12 0.20 ± 0.11 0.42 ± 0.21 * (AUC tumor/AUC plasma) (n = 7) (n = 8) (n = 5) P 5.5 ± 2.6 2.3 ± 2.7* 2.5 ± 2.9* if (mmHg) (n = 9) (n = 11) (n = 17) %collagen of dry weight 11.6 ± 5.0 5.0 ± 1.1* (n = 6) (n = 4) MDA levels 0.09 ± 0.01 0.15 ± 0.11 0.09 ± 0.02 (nmol/mg prot) (n = 5) (n = 5) (n = 5) Mean ± SD. * p < 0.05 vs control cytostatic drug, transendothelial transport, tumor P and fibrils is reduced, as shown with hydroxyproline quantifi- if the collagen fibril network. cation, this effect would not be as pronounced, and the P if might be reduced. Thus, it seems like repeated HBO treat- We found a significant reduction in tumor P after both ment have reduced tumor P by reducing collagen content if if acute and repeated HBO treatment, and expected that this and density, most likely by enhancing breakdown of col- would be reflected in an increased uptake of [ H]-5FU lagen. after both treatments. This is, however, not the fact in our study, where the uptake of [ H]-5FU was significantly Tumor vessels are hyperpermeable due to up-regulation of increased after single HBO treatment, but not after the vascular mediators such as nitric oxide, bradykinin as well repeated HBO treatment. The cpm-ratio between tumor as anatomic defects like large gap junctions between adja- and plasma was stable over the 70 min sampling-period cent cells and lack of pericytes [36,39]. An explanation for H]-5FU in the repeated HBO during microdialysis for the single HBO treatment, indi- the lack of uptake of [ cating a constant condition determining the uptake. The treated tumors, might be the proposed mechanism by Lee increased uptake of the drug into the tumor tissue, after et al. [40] of "normalization" of the abnormal structure single HBO exposure is most likely influenced by and function of the tumor vasculature after anti-ang- increased pO . Previous studies reported enhanced pO iogenic treatment. We have previously shown that 2 2 for up to one hour after HBO treatment [12,37]. Since the repeated HBO induces an anti-angiogenic effect in the repeated HBO treated animals were measured 24 hours DMBA induced tumors [19,21]. Lee et al. [40] suggested after the last HBO treatment, the pO is normalized and that it is the quality of the vascular organization and not any effect of pO will not be measurable. In the single just the quantity of the vessels that determines the vessel HBO experiments, however, the pO is still high. The function, and that the loss of endothelial cells would repeated HBO treatment probably induces more long- reduce the tortuousity of vessels or eliminate them alto- term changes in the tumor tissue that influences P . The gether. Later, several preclinical and clinical studies if reduction in collagen may be involved in the reduction of [41,42] have shown that the "normalized" vasculature P over time. Stromal fibroblasts are able to exert tension after anti-angiogenic therapy had less leaky and tortuous if on the collagen microfibrillar network through the colla- vessels, with more normal basement membranes and bet- gen binding integrins [38]. When the amount of collagen ter pericyte coverage, and that these structural changes Table 3: The extracellular volume (ECV), plasma volume (PV) and total tissue water (TTW) in skin and tumor in control and after repeated hyperbaric oxygen (HBO) exposure. ECV PV TTW (ml/g dry weight) (ml/g dry weight) (ml/g dry weight) Skin Tumor Skin Tumor Skin Tumor Control 0.55 ± 0.12 1.00 ± 0.27 0.007 ± 0.004 0.07 ± 0.04 1.44 ± 0.09 3.73 ± 0.45 (n = 9) HBO 0.50 ± 0.18 0.85 ± 0.47 0.004 ± 0.003 0.06 ± 0.03 1.45 ± 0.2 4.05 ± 0.9 (n = 8) Means ± SD. Page 6 of 9 (page number not for citation purposes) BMC Cancer 2009, 9:446 http://www.biomedcentral.com/1471-2407/9/446 Lymph vessels an Figure 2 d D) stained with LYVE-1 in two different control tumors (A and C) and hyperbaric oxygen (HBO) treated tumors (B Lymph vessels stained with LYVE-1 in two different control tumors (A and C) and hyperbaric oxygen (HBO) treated tumors (B and D). The staining was performed concomitant in the two groups. Note disintegration of lymphatics after HBO treatment, and seemingly anatomically normal lymphatics in untreated control tumors. were accompanied by "normalization" of the tumor In normal tissue, the interstitial fluid volume is kept fairly microenvironment. This modification in vascular archi- constant by several mechanisms [46], such as lymph flow tecture would decrease vascular permeability and reduce and adjustment of pressures acting across the capillary flow resistance and hence lower both mean venous pres- wall [47]. LYVE-1 surprisingly shows continuous lymph sure and P [8]. Experimental studies have demonstrated vessels in our control tumors, but dissolved lymph vessels if that anti-angiogenic therapy can decrease the overall dis- after repeated HBO treatment. However, Fukumura et al. tribution of large macromolecules such as antibodies for [48] stated that even though the structures with lymphatic instance [43,44] and decrease blood perfusion [45]. Thus, endothelial marker are present in tumors, they probably since we have previously shown that HBO has an anti- do not transport fluid or macromolecules. This, together angiogenic effect on DMBA induced tumors after repeated with the observed lowering of tumor P after repeated if HBO treatment [19], this will together with a possible HBO treatment, should lead to an increase in ECV. Never- reduced capillary permeability, impede transendothelial theless, ECV, PV and TTW did not change in tumors after transport of [ H]-5FU, even though P is lowered. repeated HBO treatment compared to control, indicating if that neither the lowered P nor the disintegrated lymphat- if ics after HBO contribute to the unchanged ECV found in Page 7 of 9 (page number not for citation purposes) BMC Cancer 2009, 9:446 http://www.biomedcentral.com/1471-2407/9/446 the present study. As already mentioned, the possible ume experiments. RKR and LS participated in the study "normalization" of the tumor vasculature, after repeated design, interpretation of data and manuscript drafting. All HBO treatment, is expected to normalize capillary perme- authors read and approved the final manuscript. ability, and thereby prevent any increase in ECV, even though P is decreased. Acknowledgements if This study was supported by grants from Helse Vest (Grants 911370), Edel and Ole Stakvold's foundation and The Norwegian cancer society. Ingrid Reactive oxygen species and oxidative stress are consid- Strand and Åse Rye Eriksen are gratefully acknowledged for technical assist- ered to be important in several aspects of malignancies, ance. both in tumor development as well as therapeutic strate- gies such as radiotherapy [49]. Since changes in oxygen References concentration may affect the production of its reactive 1. Hoogsteen IJ, Marres HA, Kogel AJ van der, Kaanders JH: The derivatives in the tumor, it is relevant to suggest that anti- hypoxic tumour microenvironment, patient selection and hypoxia-modifying treatments. Clin Oncol (R Coll Radiol) 2007, tumor effects of HBO may be due to oxidative stress. Fur- 19(6):385-396. thermore, as stated in the introduction, some 2. 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