Tissue Discs: A 3D Model for Assessing Modulation of Tissue Oxygenation.

The presence of hypoxia in solid tumours is correlated with poor treatment outcome. We have developed a 3-D tissue engineered construct to quantitatively monitor oxygen penetration through tumour tissue using the exogenous 2-nitroimidazole bioreductive probe pimonidazole and phosphorescence quenching technologies. Using this in vitro model we were able to examine the influence of the biguanides metformin and phenformin, antimycin A and KCN, on the distribution and kinetics of oxygen delivery as prototypes of modulators of oxygen metabolism.

Radiobiological effects of altering dose rate in filter-free photon beams.

To validate that altering radiotherapy dose rate through either changing pulse repetition frequency or instantaneous dose rate does not have an effect on cell survival, two human carcinoma and a hamster lung cell line were irradiated with various beam settings. Varian TrueBeam linac with a flattening filter free mode of operation was used for all experiments. The results obtained indicate that either method of changing dose rate has no effect on cell survival in the three cell lines studied. Filtered and filter free modes were also compared in treatments with protracted dose delivery which significantly increases overall treatment time. Cell survival indicated no difference between filter and filter free beam delivery in any of the protraction schemes. An increase in survival was seen in both modes upon protracting dose delivery to 15, 30 or 60 min rather than delivering acutely. Further, analysis of induced DNA double-strand breaks via the γH2AX assay showed no difference between filtered and unfiltered beams. The following study suggests that increasing dose rate is an acceptable manner of decreasing radiotherapy treatment time that does not have any detrimental effects on in vitro cell eradication.

Effects of tirapazamine on experimental colorectal liver metastases after radiofrequency ablation.

BACKGROUND: Radiofrequency ablation (RFA) is a common procedure for the management of colorectal liver metastases. RFA-generated lesions are surrounded by a rim of hypoxia that is associated with aggressive outgrowth of intrahepatic micrometastases. Hypoxia-activated prodrugs such as tirapazamine are designed selectively to induce apoptosis in tumour cells under hypoxic conditions. Therefore, it was hypothesized that tirapazamine may have therapeutic value in limiting hypoxia-associated tumour outgrowth following RFA. METHODS: Murine C26 and MC38 colorectal cancer cells were grown under hypoxia and normal oxygenation in vitro, and treated with different concentrations of tirapazamine. Apoptosis and cell cycle distribution were assessed by western blot and fluorescence-activated cell sorting analysis. Proliferative capacity was tested by means of colony-formation assays. Mice harbouring microscopic colorectal liver metastases were treated with RFA, followed by a single injection of tirapazamine (60 mg/kg) or saline. Tumour load was assessed morphometrically 7 days later. RESULTS: Tirapazamine induced apoptosis of colorectal tumour cells under hypoxia in vitro. Under normal oxygenation, tirapazamine caused a G2 cell cycle arrest from which cells recovered partly. This reduced, but did not abolish, colony-forming capacity. A single dose of tirapazamine largely prevented accelerated outgrowth of hypoxic micrometastases following RFA. Tirapazamine administration was associated with minimal toxicity. CONCLUSION: Tirapazamine induced apoptosis in colorectal cancer cells in a hypoxia-dependent manner and potently suppressed hypoxia-associated outgrowth of liver metastases with limited toxicity. This warrants further study to assess the potential value of tirapazamine, or other hypoxia-activated prodrugs, as adjuvant therapeutics following RFA treatment of colorectal liver metastases.

Metronomic gemcitabine suppresses tumour growth, improves perfusion, and reduces hypoxia in human pancreatic ductal adenocarcinoma.

BACKGROUND: The current standard of care for pancreatic cancer is weekly gemcitabine administered for 3 of 4 weeks with a 1-week break between treatment cycles. Maximum tolerated dose (MTD)-driven regimens as such are often associated with toxicities. Recent studies demonstrated that frequent dosing of chemotherapeutic drugs at relatively lower doses in metronomic regimens also confers anti-tumour activity but with fewer side effects. METHODS: Herein, we evaluated the anti-tumour efficacy of metronomic vs MTD gemcitabine, and investigated their effects on the tumour microenvironment in two human pancreatic cancer xenografts established from two different patients. RESULTS: Metronomic and MTD gemcitabine significantly reduced tumour volume in both xenografts. However, K(trans) values were higher in metronomic gemcitabine-treated tumours than in their MTD-treated counterparts, suggesting better tissue perfusion in the former. These data were further supported by tumour-mapping studies showing prominent decreases in hypoxia after metronomic gemcitabine treatment. Metronomic gemcitabine also significantly increased apoptosis in cancer-associated fibroblasts and induced greater reductions in the tumour levels of multiple pro-angiogenic factors, including EGF, IL-1alpha, IL-8, ICAM-1, and VCAM-1. CONCLUSION: Metronomic dosing of gemcitabine is active in pancreatic cancer and is accompanied by pronounced changes in the tumour microenvironment.

The effect of deep inspiration breath-hold on tumour oxygenation.

AIM: To investigate the influence of deep inspiration breath-hold on the oxygen tension of in-vivo tumours measured using an Eppendorf pO2 histograph. MATERIALS AND METHODS: Patients with accessible primary or metastatic tumours > or = 2 cm diameter were entered into a protocol measuring tumour oxygenation with an Eppendorf pO2 histograph during normal breathing (NB) and deep inspiration breath-hold (DIBH). Change in oxygen tension was assessed using the Wilcoxon Signed Ranks test. RESULTS: Thirty patients were entered in to this protocol. The median maximum tumour dimension was 4 cm. The median of the median pO2 of these tumours was 18 mmHg. Tumours were assessed during NB and DIBH. Oxygen tension measurements along 1-3 pairs of tracks per tumour (median of 2) were obtained. The median number of measurements per track was 30 for NB and 29 for DIBH (range 17-59). In six tumours, the values during NB were significantly higher than during DIBH, whereas, for six other tumours, the relationship was the opposite; for the remaining 18 patients, no significant difference was observed. CONCLUSION: These data show heterogeneity of tumour oxygenation seen with in-situ tumours both at baseline and as a result of DIBH. No systematic change in the Eppendorf pO2 measurements was seen as a result of DIBH; however, the individual tumour responses to DIBH varied dramatically.

Oxygen tension in primary gynaecological tumours: the influence of carbon dioxide concentration.

BACKGROUND AND PURPOSE: To assess the effect of inhalation of various high oxygen content gases (HOCG) with different carbon dioxide concentrations on the tumour oxygen tension in patients with primary gynaecological malignancies. MATERIALS AND METHODS: Tumour oxygen tension was assessed on two protocols in those patients with locally advanced visible or palpable primary gynaecological malignancies. Patients were assessed initially while breathing room air (R/A). After 4 min of inhaling the first HOCG, a second assessment of the oxygen tension within the tumour was made. After a 10 min rest period while inhaling R/A, the second HOCG was administered for 4 min after which the third set of measurements were obtained. Protocol A involved assessing the tumour oxygen tension in 12 patients while breathing R/A, 100% oxygen (O(2)) and 5% carbogen (95% O(2), 5% CO(2)). For protocol B, tumour oxygen tension assessments of 13 patients while breathing R/A, 2.5% carbogen (97.5% O(2), 2.5% CO(2)), and 5% carbogen. Median pO(2) and percentage of values

Carbogen inhalation in cervical cancer: assessment of oxygenation change.

OBJECTIVE: Our objectives were (1) to examine tumor oxygenation measured with an Eppendorf pO(2) histograph, prior to and during carbogen (95% oxygen, 5% carbon dioxide) breathing in patients with primary cervical cancer; and (2) to assess the feasibility of delivering external beam radiation therapy and concurrent carbogen to patients treated for cervical cancer. METHODS: Pretreatment tumoral pO(2) measurements were obtained using an Eppendorf pO(2) histograph in patients with primary cervical cancers while breathing room air and after 4 min of carbogen breathing. Patients able to tolerate the carbogen inhalation were asked to inhale it for 4 min prior to and during all external beam radiation therapy. RESULTS: Two sets of pO(2) measurements were obtained from 25 patients. The average median pO(2) increased from 8 mm Hg when breathing room air to 96 mm Hg after carbogen breathing. Twenty-four of 25 patients tolerated the carbogen; they inhaled carbogen during their daily external beam radiation therapy. All 24 patients completed their planned course of external beam radiation therapy and daily concurrent carbogen without significant difficulty. CONCLUSION: (1) Carbogen inhalation increased the average median pO(2) value 10-fold and decreased the percentage of values

Characterization of three-dimensional tissue cultures using electrical impedance spectroscopy.

Electrical impedance spectroscopy was used to characterize the cell environment of multilayered cell cultures (MCCs), a culture system in which cells are grown on a permeable support membrane to form a thick disc of cells with tumor-like properties. Cultures were grown using SiHa tumor cells as well as V79 wild-type cells and V79/DOX cells cultivated to exhibit multidrug resistance. Electrical impedance measurements were made on MCCs over a frequency range of 0. 1 kHz to 1 MHz. Data analysis using a simple electrical model for the cell environment yielded estimates for parameters related to the intra- and extracellular resistance and net membrane capacitance, which were then related to MCC thickness. The extracellular fraction and tortuosity of the MCCs were determined in separate experiments where the rate of diffusion and the equilibrium level of C14-inulin, which does not penetrate the cell membrane, was measured within MCCs. Impedance measurements predicted the barrier to diffusion posed by the extracellular space of MCCs to be roughly two times greater than that inferred from the C14-inulin experiments. However, the relative ranking of the three cell types used to grow MCCs was similar for the two methods. Results indicate that impedance spectroscopy is well suited for use in characterizing the MCC cell environment, offering a fast, nondestructive method for monitoring cell culture growth and integrity.

Measurement of delivery and metabolism of tirapazamine to tumour tissue using the multilayered cell culture model.

PURPOSE: Efficient extravascular penetration is essential for the optimal activity of most anticancer drugs and is particularly relevant to bioreductive cytotoxins which target hypoxic cells that can be located distal to functional blood vessels within tumours. Tirapazamine (3-amino-1,2,4-benzotriazine-1,4-di-N-oxide; Triazone; SR 259075; formerly SR 4233) is a lead bioreductive cytotoxin currently undergoing clinical evaluation. It exhibits preferential cytotoxicity towards cells at reduced oxygen tension, and could complement existing anticancer therapies where hypoxic cells are believed to constitute a refractory population. We assessed the ability of tirapazamine to penetrate tumour tissue using an in vitro multilayered cell culture (MCC) model. METHODS: Diffusion of tirapazamine through oxic and hypoxic multilayered cell cultures composed of SiHa. human cervical carcinoma cells, was measured using a dual reservoir diffusion apparatus from which samples were quantified via HPLC. Drug concentration kinetics from both reservoirs were analysed using a mathematical model for diffusion and metabolism within the MCC. Results were then applied to a second mathematical model which described extravascular drug penetration within a tumour cord, the sheath of cells surrounding a blood vessel. RESULTS: The diffusion coefficient of tirapazamine within SiHa MCCs was determined as 7.0+/-0.5 x 10(-7) cm2/s and the maximal metabolic rate for hypoxic MCCs, Vmax, as 1.5+/-0.4 microM/s. The thickness of individual tissue cultures was determined by diffusion of tritiated water (HTO). A linear relationship was shown to exist between tissue thickness and the inverse of permeability to HTO. Experimental results were used to simulate drug distribution within a tumour cord. These simulations indicate that, when tirapazamine is administered via intravenous infusion, a stable tirapazamine distribution throughout the cord occurs within 15 min with cells most peripheral to the blood vessel exposed to only 10% of the blood drug concentration. Under these conditions, the simulations predict cell kill to be limited to the first 75 microm of tissue surrounding a blood vessel. CONCLUSION: This study indicates that extravascular penetration of tirapazamine to peripheral cells existing at low oxygen tension may be limited by the metabolism of tirapazamine by more proximal cells existing at moderate oxygen tension. Simulations found that tirapazamine reached only 10% of the blood concentration at cells most peripheral to blood vessels. These results indicate that tirapazamine would be significantly cytotoxic only to cells located within approximately 75 microm of blood vessels. Further MCC-based modelling of extravascular drug penetration would serve as a means of identifying new antitumour agents with location-specific activity.

The relative biological effectiveness of the modulated proton beam at TRIUMF.

PURPOSE: The purpose of this study was to determine the relative biological effectiveness (RBE) of proton beam and to study its dependency on fraction number. METHODS: The relative biological effectiveness (RBE) of fractionated protons compared with 60Co gamma-rays was investigated for the acute mouse skin reaction. The 80 MeV protons generated at the TRIUMF cyclotron were spread out from 7 to 25 mm to irradiate entire legs. One, two, four, and eight fractions were tested. RESULTS: RBE values ranged from 1.15 to 1.24 for the acute skin reaction. Fraction dose dependence of RBEs was not observed.

Multilayers of cells growing on a permeable support. An in vitro tumour model.

A system for growing three-dimensional cell culture has been developed which exhibits many features of solid tumours. This system comprises cells growing as a thick mat on a semipermeable membrane suspended in stirred media. SiHa cells grown as these multilayered cell cultures (MCCs) have produced cultures up to ca. 20 cell diameters in thickness. The MCCs, like solid tumours growing in vivo. develop diffusion-dependent necrosis and hypoxia and the cell packing acts as a barrier to the diffusion of drugs. These cultures can, therefore, be used to study aspects of cancer biology and drug transport that are difficult to study using other techniques.

The effect of thalidomide on experimental tumors and metastases.

Thalidomide has recently been shown to antagonize basic fibroblast growth factor-induced angiogenesis in the rat corneal micropocket assay. We have investigated the effect of thalidomide on growth, radiosensitivity and metastasis in murine SCCVII and Lewis Lung tumors. We found that daily thalidomide administration (0.77 mmol/kg/day, i.p.) does not alter primary tumor growth of SCCVII or Lewis Lung tumors. However, thalidomide administration does reduce radiosensitivity of the Lewis Lung tumor, and increases its sensitivity to combined treatment with radiation and the bioreductive cytotoxin tirapazamine. These findings suggest that thalidomide is elevating tumor hypoxia in the Lewis Lung tumor, presumably via an anti-angiogenic mechanism. We also found that thalidomide administration reduces the incidence of lung metastases from primary Lewis Lung tumors. Thalidomide may therefore have utility in the management of solid tumors, especially when combined with drugs that are selectively toxic to cells at reduced oxygen tension (e.g. bioreductive cytotoxins).

Cytotoxic effect of RB 6145 in human tumour cell lines: dependence on hypoxia, extra- and intracellular pH and drug uptake.

Low pH and hypoxia are a common feature of many solid tumours. This study examined the effect of these two conditions on the cytotoxic properties of the bifunctional agent RB 6145, the prodrug of RSU 1069. The effect of acidic pH on RB 6145 toxicity was examined in six human tumour cell lines under hypoxic conditions and was found to have little effect in HT 29, A549, U373 and HT 144 cells. Treatment was for 1 h at 37 degrees C, pH 6.4 or 7.4. Significant potentiation of RB 6145 toxicity was observed in SiHa cells (enhancement ratio; ERpH approximately 1.6) and in U1 cells (ERpH approximately 1.4). In these two cell lines the potentiation of RB 6145 toxicity arising from hypoxia was large, with ERHyp approximately 11 and 15 in SiHa and U1 cells respectively. SiHa cells, which show a pH effect and HT 29 cells, which do not, were chosen for further comparative studies of drug uptake )nd regulation of intracellular pH. High-performance liquid chromatography (HPLC) determinations of the uptake of RB 6145 and its dervatives showed that in SiHa cells, intracellular to extracellular drug concentration ratio (Ci/Ce) at 1 h was approximately 40% higher at pH 6.4 than at pH 7.4, whereas in HT 29 cells Ci/Ce was approximately 25% lower. Under conditions of acidic extracellular pH, regulation of pH was somewhat less effective in SiHa cells, where pHi dropped to within 0.2 pH units of the extracellular pH over a 2.5 h treatment at pH 6.4. It seems likely that increased drug uptake was at least part of the basis for the observed potentiation of RB 6145 toxicity in SiHa cells. A model which would better explain the results for both cell lines might also include the possibility that low pH per se potentiates cytotoxic damage to a modest extent and that it is offset or augmented by altered uptake in HT 29 and SiHa cells respectively.

Computerized histographic characterization of changes in tissue pO2 induced by erythropoietin.

Anemia associated with malignancy is a common clinical problem, and has a negative effect on oxygen delivery and, therefore, response of tumors to radiotherapy. Erythropoietin (EPO) has been shown to increase the hematocrit of rodents when administered subcutaneously. The objectives of this study were to measure tumor and normal tissue pO2 with computerized pO2 histography, to characterize the change in rodent hematocrit with EPO administration, and to assess the capacity of pO2 histography to measure changes in tumor pO2 during growth, with or without EPO administration. Ten C3H mice were implanted with SCCVII tumors and after three weeks of tumor growth, EPO (1500 IU/kg/day) was administered for two weeks. Tumor and subcutaneous (SQ) measurements were made weekly with an Eppendorf pO2 histograph and hematocrits were obtained concomitantly. Eight C3H/SCCVII mice, which received no EPO and underwent similar measurements, served as controls. The hematocrits of the control mice dropped progressively from 42.0 to 23.0% during the two week period. There was a corresponding fall in both the SQ and tumor mean pO2 (55.6 to 40.3 mm Hg, and 19.9 to 10.0 mm Hg, respectively). In the treated group, the hematocrits remained stable (38.0 to 43.1%) as did the mean pO2 of the SQ (46.1 to 54.3 mm Hg) and the tumor (11.1 to 11.3 mm Hg). These data lend support to the value of EPO in reversing the anemia associated with malignancy and suggest a role of pO2 histography in monitoring the beneficial effects of EPO therapy.

The hypoxic cytotoxin SR 4233 increases the effectiveness of radioimmunotherapy in mice with human non-Hodgkin's lymphoma xenografts.

PURPOSE: To determine if either the hypoxic cell radiosensitizer etanidazole (SR 2508) or the hypoxic cytotoxin SR 4233 could improve the effectiveness of radioimmunotherapy. METHODS AND MATERIALS: LC4 (an IgG1 monoclonal antibody directed toward malignant T cells) and MB-1 (an irrelevant isotype-matched control antibody) were injected intraperitoneally into severe combined immunodeficient phenotype mice with human cutaneous T cell lymphoma xenografts in order to determine the distribution of the antibodies in the tumors and normal tissues as a function of time. Computerized-pO2-histography was used to measure the median oxygen tension in the tumors. Tumor-bearing mice were treated with: (a) LC4; (b) 90Y-LC4; (c) 90Y-MB-1; (d) whole body irradiation delivered via an external 137Cs source; (e) etanidazole and 90Y-LC4; (f) SR 4233 and 90Y-LC4; (g) etanidazole; and (h) SR 4233. An additional group of mice received no treatment and served as controls. A tumor growth delay assay was used to assess the effectiveness of the different treatment regimens. RESULTS: LC4 accumulated in the tumors to a significantly greater extent than MB-1 (p < 0.001) and reached a peak concentration in the tumors 5 days post-injection. The human cutaneous T cell lymphoma xenografts had a relatively low median oxygen tension. LC4 by itself was able to produce a minor decrease in tumor size (control vs. LC4; p = 0.001). 90Y-LC4 produced greater tumor growth delay than LC4 alone (LC4 vs. 90Y-LC4; p = 0.01); however, the Yttrium-90 caused neutropenia and weight loss. The 90Y-labeled tumor-specific and non-specific antibodies both exerted greater tumor growth delay than externally delivered whole body irradiation (p < or = 0.03) due to preferential uptake of the antibodies in the tumors. Etanidazole and SR 4233 by themselves did not significantly inhibit the growth of the tumors. Etanidazole did not significantly enhance the tumor growth delay produced by 90Y-LC4 (90Y-LC4 vs etanidazole and 90Y-LC4, p = 0.13). SR 4233, on the other hand, did enhance the tumor growth delay produced by 90Y-LC4 (90Y-LC4 vs. SR 4233 and 90Y-LC4, p = 0.046). The neutropenia and weight loss caused by 90Y-LC4 were exacerbated slightly (< 10%) by the administration of SR 4233. CONCLUSIONS: A first generation hypoxic cytotoxin, SR 4233, was able to enhance the tumor growth delay produced by radioimmunotherapy in severe combined immunodeficient phenotype mice with human cutaneous T cell lymphoma xenografts.

Enhancement of the cytotoxicity of SR 4233 to normal and malignant tissues by hypoxic breathing.

The bioreductive cytotoxic agent SR 4233 (1,2,4-benzotriazine 3-amine 1,4-dioxide) has been shown to markedly potentiate the cell killing of mouse tumours when combined with fractionated radiation therapy. Differential metabolism under oxic compared to hypoxic conditions results in SR 4233 exhibiting selective cytotoxicity to hypoxic cells. This is thought to result from the production of a cytotoxic free radical which is generated predominantly in the absence of oxygen. We have examined a way of enhancing the effectiveness of this antitumour agent in vivo by artificially increasing the hypoxic fraction of tumours by hypoxic breathing. Mice are placed in a chamber containing 10% Oxygen 90% Nitrogen for 1 h after each administration of SR 4233. Our results in the SCCVII tumour model indicate that this manoeuvre results in a 10-fold increase in antitumour effectiveness of SR 4233 when administered in a fractionated regime with radiotherapy (8 x 2.5 Gy and 0.08 mmol kg-1), but not when a single treatment regime (1 x 20 Gy and 0.3 mmol kg-1) is used. Mathematical modelling of this effect is used to illustrate this phenomenon and can be used to predict the dependence of this type of therapy on the modification of tumour oxygenation.

Characterization of a CHO cell line resistant to killing by the hypoxic cell cytotoxin SR 4233.

One approach to understanding the mechanism of selective hypoxic cell killing by the benzotriazine-di-N-oxide, SR 4233, is to characterize cell lines that exhibit increased resistance to killing by this drug. The Chinese Hamster Ovary cell line BL-10 was originally isolated on the basis of its hypersensitivity to killing by bleomycin. It is 2.7-fold more resistant to hypoxic cell killing by SR 4233 than wild-type CHO on comparison of D0's. However, both BL-10 and CHO possess the same sensitivity to killing by SR 4233 under aerobic conditions. We have excluded the explanation that differential metabolism of SR 4233 is responsible for its increased survival as both BL-10 and CHO produce the two-electron product SR 4317 at the same rate (3 nmoles/hr/10(6) cells). Analysis of free radical production, DNA double-strand break induction, and glutathione (GSH) levels suggested that the resistance of BL-10 to killing by SR 4233 might result from increased intracellular radical scavenger pathways. Using buthionine sulfoximine (BSO) to decrease cellular GSH levels, we found a marked increase in the sensitivity of BL-10 cells to SR 4233 killing under hypoxia, but a much smaller increase in the sensitivity of CHO cells. Taken together, these data imply that the high GSH levels in BL-10 cells is responsible for its resistance to SR 4233 cytotoxicity.

Second-generation 1,2,4-benzotriazine 1,4-di-N-oxide bioreductive anti-tumor agents: pharmacology and activity in vitro and in vivo.

SR 4233 (1,2,4-benzotriazine-3-amine 1,4-dioxide) will soon be entering Phase I clinical trials as a new bioreductive cytotoxic agent for the treatment of solid tumors in combination with fractionated radiotherapy. We have selected 3 from over 50 analogues of SR 4233 which showed particular promise as second generation bioreductive antitumor agents. These compounds, when compared to SR 4233, have higher hypoxic toxicity and comparable or higher oxic to hypoxic cytotoxicity ratios in vitro and similar animal toxicity. We have compared the effectiveness of these three compounds with SR 4233 in two tumor systems and have examined some pharmacokinetic properties. The results show that replacement of the amino group at the 3-position of SR 4233 with either a hydrogen or an N,N-dialkylaminoalkylamino group shortens the half-life of these compounds in the blood because of the combined effects of partition coefficients, basicity, and higher reactivity. SR 4754 and SR 4755, the N,N-dialkylaminoalkylamino derivatives, exhibited shorter plasma half-lives than SR 4233 but exhibited lower anti-tumor activity than SR 4233 based on equal mouse toxicity in a fractionated regimen. SR 4482, with the hydrogen substitution and very high electron affinity, possessed a very short blood half life yet retained similar anti-tumor activity as SR 4233.