GH3 Prolactinomas Research Articles

Evaluation and Immunohistochemical Qualification of Carbogen-Induced ΔR2* as a Noninvasive Imaging Biomarker of Improved Tumor Oxygenation

To evaluate and histologically qualify carbogen-induced ΔR2 as a noninvasive magnetic resonance imaging biomarker of improved tumor oxygenation using a double 2-nitroimidazole hypoxia marker approach. Multigradient echo images were acquired from mice bearing GH3 prolactinomas, preadministered with the hypoxia marker CCI-103F, to quantify tumor R2 during air breathing. With the mouse remaining positioned within the magnet bore, the gas supply was switched to carbogen (95% O2, 5% CO2), during which a second hypoxia marker, pimonidazole, was administered via an intraperitoneal line, and an additional set of identical multigradient echo images acquired to quantify any changes in tumor R2. Hypoxic fraction was quantified histologically using immunofluorescence detection of CCI-103F and pimonidazole adduct formation from the same whole tumor section. Carbogen-induced changes in tumor pO2 were further validated using the Oxylite fiberoptic probe. Carbogen challenge significantly reduced mean tumor R2 from 116 ± 13 s(-1) to 97 ± 9 s(-1) (P<.05). This was associated with a significantly lower pimonidazole adduct area (2.3 ± 1%), compared with CCI-103F (6.3 ± 2%) (P<.05). A significant correlation was observed between ΔR2 and Δhypoxic fraction (r=0.55, P<.01). Mean tumor pO2 during carbogen breathing significantly increased from 6.3 ± 2.2 mm Hg to 36.0 ± 7.5 mm Hg (P<.01). The combined use of intrinsic susceptibility magnetic resonance imaging with a double hypoxia marker approach corroborates carbogen-induced ΔR2 as a noninvasive imaging biomarker of increased tumor oxygenation.

Exploring ΔR2* and ΔR1 as imaging biomarkers of tumor oxygenation

To investigate the combined use of hyperoxia-inducedΔR(2) * and ΔR(1) as a noninvasive imaging biomarker of tumor hypoxia. MRI was performed on rat GH3 prolactinomas (n = 6) and human PC3 prostate xenografts (n = 6) propagated in nude mice. multiple gradient echo and inversion recovery truefisp images were acquired from identical transverse slices to quantify tumor R(2) * and R(1)before and during carbogen (95% O2 /5% CO2 ) challenge, and correlates of ΔR(2) * and ΔR(1) assessed. Mean baseline R(2) * and R(1) were 119 ± 7 s(-1) and 0.6 ± 0.03 s(-1) for GH3 prolactinomas and 77 ± 12 s(-1) and 0.7 ± 0.02 s(-1) for PC3 xenografts, respectively. During carbogen breathing, mean ΔR(2) * and ΔR(1) were -20 ± 8 s(-1) and 0.08 ± 0.03 s(-1) for GH3 and -0.5 ± 1 s(-1) and 0.2 ± 0.08 s(-1) for the PC3 tumors, respectively. A pronounced relationship betweenΔR(2) * and ΔR(1) was revealed. Considering the blood oxygen-hemoglobin dissociation curve, fast R2 * suggested that GH3 prolactinomas were more hypoxic at baseline, and their carbogen response dominated by increased hemoglobin oxygenation, evidenced by highly negative ΔR(2) *. PC3 tumors were less hypoxic at baseline, and their response to carbogen dominated by increased dissolved oxygen, evidenced by highly positive ΔR(1) . Because the two biomarkers are sensitive to different oxygenation ranges, the combination of ΔR(2) * and ΔR(1) may better characterize tumor hypoxia than each alone.

Assessment of Tumor Response to the Vascular Disrupting Agents 5,6-Dimethylxanthenone-4-Acetic Acid or Combretastatin-A4-Phosphate by Intrinsic Susceptibility Magnetic Resonance Imaging

To investigate the use of the transverse magnetic resonance imaging (MRI) relaxation rate R(2)(*) (s(-1)) as a biomarker of tumor vascular response to monitor vascular disrupting agent (VDA) therapy. Multigradient echo MRI was used to quantify R(2)(*) in rat GH3 prolactinomas. R(2)(*) is a sensitive index of deoxyhemoglobin in the blood and can therefore be used to give an index of tissue oxygenation. Tumor R(2)(*) was measured before and up to 35 min after treatment, and 24 h after treatment with either 350 mg/kg 5,6-dimethylxanthenone-4-acetic acid (DMXAA) or 100 mg/kg combretastatin-A4-phosphate (CA4P). After acquisition of the MRI data, functional tumor blood vessels remaining after VDA treatment were quantified using fluorescence microscopy of the perfusion marker Hoechst 33342. DMXAA induced a transient, significant (p < 0.05) increase in tumor R(2)(*) 7 min after treatment, whereas CA4P induced no significant changes in tumor R(2)(*) over the first 35 min. Twenty-four hours after treatment, some DMXAA-treated tumors demonstrated a decrease in R(2)(*), but overall, reduction in R(2)(*) was not significant for this cohort. Tumors treated with CA4P showed a significant (p < 0.05) reduction in R(2)(*) 24 h after treatment. The degree of Hoechst 33342 uptake was associated with the degree of R(2)(*) reduction at 24 h for both agents. The reduction in tumor R(2)(*) or deoxyhemoglobin levels 24 h after VDA treatment was a result of reduced blood volume caused by prolonged vascular collapse. Our results suggest that DMXAA was less effective than CA4P in this rat tumor model.

Susceptibility Contrast Magnetic Resonance Imaging Determination of Fractional Tumor Blood Volume: A Noninvasive Imaging Biomarker of Response to the Vascular Disrupting Agent ZD6126

To assess tumor fractional blood volume (xi), determined in vivo by susceptibility contrast magnetic resonance imaging (MRI) as a noninvasive imaging biomarker of tumor response to the vascular disrupting agent ZD6126. The transverse MRI relaxation rate R(2)( *) of rat GH3 prolactinomas was quantified prior to and following injection of 2.5 mgFe/kg feruglose, an ultrasmall superparamagnetic iron oxide intravascular contrast agent, and xi (%) was determined from the change in R(2)( *). The rats were then treated with either saline or 50 mg/kg ZD6126, and xi measured again 24 hours later. Following posttreatment MRI, Hoechst 33342 (15 mg/kg) was administered to the rats and histological correlates from composite images of tumor perfusion and necrosis sought. Irrespective of treatment, tumor volume significantly increased over 24 hours. Saline-treated tumors showed no statistically significant change in xi, whereas a significant (p = 0.002) 70% reduction in xi of the ZD6126-treated cohort was determined. Hoechst 33342 uptake was associated with viable tumor tissue and was significantly (p = 0.004) reduced and restricted to the rim of the ZD6126-treated tumors. A significant positive correlation between posttreatment xi and Hoechst 33342 uptake was obtained (r = 0.83, p = 0.002), providing validation of the MRI-derived measurements of fractional tumor blood volume. These data clearly highlight the potential of susceptibility contrast MRI with ultrasmall superparamagnetic iron oxide contrast agents to provide quantitative imaging biomarkers of fractional tumor blood volume at high spatial resolution to assess tumor vascular status and response to vascular disrupting agents.

Rat Tumor Response to the Vascular-Disrupting Agent 5,6-Dimethylxanthenone-4-Acetic Acid as Measured by Dynamic Contrast-Enhanced Magnetic Resonance Imaging, Plasma 5-Hydroxyindoleacetic Acid Levels, and Tumor Necrosis

The dose-dependent effects of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) on rat GH3 prolactinomas were investigated in vivo. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used to assess tumor blood flow/permeability pretreatment and 24 hours posttreatment with 0, 100, 200, or 350 mg/kg DMXAA. DCE-MRI data were analyzed using Ktrans and the integrated area under the gadolinium time curve (IAUGC) as response biomarkers. Highperformance liquid chromatography (HPLC) was used to determine the plasma concentration of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) following treatment to provide an index of increased vessel permeability and vascular damage. Finally, tumor necrosis was assessed by grading hematoxylin and eosin-stained sections cut from the same tumors investigated by MRI. Both tumor Ktrans and IAUGC were significantly reduced 24 hours posttreatment with 350 mg/kg DMXAA only, with no evidence of dose response. HPLC demonstrated a significant increase in plasma 5-HIAA 24 hours posttreatment with 200 and 350 mg/kg DMXAA. Histologic analysis revealed some evidence of tumor necrosis following treatment with 100 or 200 mg/kg DMXAA, reaching significance with 350 mg/kg DMXAA. The absence of any reduction in Ktrans or IAUGC following treatment with 200 mg/kg, despite a significant increase in 5-HIAA, raises concerns about the utility of established DCE-MRI biomarkers to assess tumor response to DMXAA.

Acute Tumor Response to ZD6126 Assessed by Intrinsic Susceptibility Magnetic Resonance Imaging

The effective magnetic resonance imaging (MRI) transverse relaxation rate R2* was investigated as an early acute marker of the response of rat GH3 prolactinomas to the vascular-targeting agent, ZD6126. Multigradient echo (MGRE) MRI was used to quantify R2*, which is sensitive to tissue deoxyhemoglobin levels. Tumor R2* was measured prior to, and either immediately for up to 35 minutes, or 24 hours following administration of 50 mg/kg ZD6126. Following MRI, tumor perfusion was assessed by Hoechst 33342 uptake. Tumor R2* significantly increased to 116 ± 4% of baseline 35 minutes after challenge, consistent with an ischemic insult induced by vascular collapse. A strong positive correlation between baseline R2* and the subsequent increase in R2* measured 35 minutes after treatment was obtained, suggesting that the baseline R2* is prognostic for the subsequent tumor response to ZD6126. In contrast, a significant decrease in tumor R2* was found 24 hours after administration of ZD6126. Both the 35-minute and 24-hour R2* responses to ZD6126 were associated with a decrease in Hoechst 33342 uptake. Interpretation of the R2* response is complex, yet changes in tumor R2* may provide a convenient and early MRI biomarker for detecting the antitumor activity of vascular-targeting agents.

In vivo determination of tumor oxygenation during growth and in response to carbogen breathing using 15C5-loaded alginate capsules as fluorine-19 magnetic resonance imaging oxygen sensors

<h3>Purpose</h3> The objective was to present a method for the repeated noninvasive measurement of tumor oxygenation (Po₂) over the whole period of tumor growth. <h3>Methods and materials</h3> A mixture of tumor homogenate (GH3 prolactinoma) and alginate capsules loaded with perfluoro-15-crown-5-ether (15C5) was injected into the flanks of Wistar Furth rats. The temporal behavior of tumor Po₂ was monitored between Day 1 and 26 after injection using fluorine-19 (¹⁹F) magnetic resonance imaging (MRI). In addition, the response of tumor Po₂ to modifiers of the tumor microenvironment (carbogen [95% O₂/5% CO₂], nicotinamide, and hydralazine) was investigated. <h3>Results</h3> An initial increase of tumor Po₂, probably reflecting neovascularization, followed by a decrease after Week 2, probably indicating tumor hypoxia or necrosis, were observed. The minimum and maximum average Po₂ ± SEM observed were 3.3 ± 2.0 mm Hg on Day 2 and 25.7 ± 3.8 mm Hg on Day 13, respectively. Carbogen increased the tumor Po₂, whereas nicotinamide caused no significant change and hydralazine induced a significant decrease in tumor oxygenation. <h3>Conclusions</h3> A preclinical method for the repeated noninvasive determination of tumor Po₂ was presented. It might help to investigate tumor physiology and the mechanisms of modifiers of the tumor microenvironment and their role in different therapeutic approaches.

Investigations in vivo of the effects of carbogen breathing on 5-fluorouracil pharmacokinetics and physiology of solid rodent tumours

We have shown previously that carbogen (95% 0(2), 5% CO(2)) breathing by rodents can increase uptake of anticancer drugs into tumours. The aim of this study was to extend these observations to other rodent models using the anticancer drug 5-fluorouracil (5FU). 5FU pharmacokinetics in tumour and plasma and physiological effects on the tumour by carbogen were investigated to determine the locus of carbogen action on augmenting tumour uptake of 5FU. Two different tumour models were used, rat GH3 prolactinomas xenografted s.c. into nude mice and rat H9618a hepatomas grown s.c. in syngeneic Buffalo rats. Uptake and metabolism of 5FU in both tumour models with or without host carbogen breathing was studied non-invasively using fluorine-19 magnetic resonance spectroscopy ((19)F-MRS), while plasma samples from Buffalo rats were used to construct a NONMEM pharmacokinetic model. Physiological effects of carbogen on tumours were studied using (31)P-MRS for energy status (NTP/Pi) and pH, and gradient-recalled echo magnetic resonance imaging (GRE-MRI) for blood flow and oxygenation. In both tumour models, carbogan-induced GRE-MRI signal intensity increases of approximately 60% consistent with an increase in tumour blood oxygenation and/or flow. In GH3 xenografts, (19)F-MRS showed that carbogen had no significant effect on 5FU uptake and metabolism by the tumours, and (31)P-MRS showed there was no change in the NTP/Pi ratio. In H9618a hepatomas, (19)F-MRS showed that carbogen had no effect on tumour 5FU uptake but significantly ( p=0.0003) increased 5FU elimination from the tumour (i.e. decreased the t(1/2)) and significantly ( p=0.029) increased (53%) the rate of metabolism to cytotoxic fluoronucleotides (FNuct). The pharmacokinetic analysis showed that carbogen increased the rate of tumour uptake of 5FU from the plasma but also increased the rate of removal. (31)P-MRS showed there were significant ( p<or=0.02) increases in the hepatoma NTP/Pi ratio of 49% and transmembrane pH gradient of 0.11 units. We suggest that carbogen can transiently increase tumour blood flow, but this effect alone may not increase uptake of anticancer drugs without a secondary mechanism operating. In the case of the hepatoma, the increase in tumour energy status and pH gradient may be sufficient to augment 5FU metabolism to cytotoxic FNuct, while in the GH3 xenografts this was not the case. Thus carbogen breathing does not universally lead to increased uptake of anticancer drugs.

In vivo determination of tumor oxygenation during growth and in response to carbogen breathing using 15C5-loaded alginate capsules as fluorine-19 magnetic resonance imaging oxygen sensors.

The objective was to present a method for the repeated noninvasive measurement of tumor oxygenation (Po(2)) over the whole period of tumor growth. A mixture of tumor homogenate (GH3 prolactinoma) and alginate capsules loaded with perfluoro-15-crown-5-ether (15C5) was injected into the flanks of Wistar Furth rats. The temporal behavior of tumor Po(2) was monitored between Day 1 and 26 after injection using fluorine-19 ((19)F) magnetic resonance imaging (MRI). In addition, the response of tumor Po(2) to modifiers of the tumor microenvironment (carbogen [95% O(2)/5% CO(2)], nicotinamide, and hydralazine) was investigated. An initial increase of tumor Po(2), probably reflecting neovascularization, followed by a decrease after Week 2, probably indicating tumor hypoxia or necrosis, were observed. The minimum and maximum average Po(2) +/- SEM observed were 3.3 +/- 2.0 mm Hg on Day 2 and 25.7 +/- 3.8 mm Hg on Day 13, respectively. Carbogen increased the tumor Po(2), whereas nicotinamide caused no significant change and hydralazine induced a significant decrease in tumor oxygenation. A preclinical method for the repeated noninvasive determination of tumor Po(2) was presented. It might help to investigate tumor physiology and the mechanisms of modifiers of the tumor microenvironment and their role in different therapeutic approaches.

Tumor R2* is a prognostic indicator of acute radiotherapeutic response in rodent tumors.

To test the prognostic potential of tumor R2* with respect to radiotherapeutic outcome. Blood oxygenation level dependent (BOLD) MRI images are sensitive to changes in deoxyhemoglobin concentration through the transverse MRI relaxation rate R2* of tissue water, hence the quantitative measurement of tumor R2* may be related to tissue oxygenation. Tumor growth inhibition in response to radiation was established for both GH3 prolactinomas and RIF-1 fibrosarcomas with animals breathing either air or carbogen during radiation. In a separate cohort, the baseline R2* and carbogen (95% O2, 5% CO2)-induced DeltaR2* of rat GH3 prolactinomas and murine RIF-1 fibrosarcomas were quantified using multigradient echo (MGRE) MRI prior to radiotherapy, and correlated with subsequent tumor growth inhibition in response to ionizing radiation, while the animals breathed air. A radiation dose of 15 Gy caused pronounced growth delay in both tumor models and transient regression of the GH3 prolactinomas. When the animals breathed carbogen during radiation, the growth delay/regression was enhanced only in the GH3 prolactinomas. The GH3 prolactinomas, which exhibit a relatively fast baseline R2* and large DeltaR2* in response to carbogen breathing prior to radiotherapy, showed a substantial reduction in normalized tumor volume to 66 +/- 3% with air breathing and 36 +/- 5% with carbogen seven days after 15 Gy irradiation. In contrast, the effect of 15 Gy on the RIF-1 fibrosarcomas, which give a relatively slow baseline R2* and negligible DeltaR2* response to carbogen prior to treatment, showed a much smaller growth inhibition (143 +/- 3% with air, 133 +/- 12% with carbogen). Quantitation of tumor R2* and carbogen-induced DeltaR2* by MGRE MRI provides completely noninvasive prognostic indicators of a potential acute radiotherapeutic response.

Single Dose of the Antivascular Agent, ZD6126 (N-Acetylcoichinol-O-Phosphate), Reduces Perfusion for at Least 96 Hours in the GH3 Prolactinoma Rat Tumor Model

Tumor vasculature is an attractive therapeutic target as it differs structurally from normal vasculature, and the destruction of a single vessel can lead to the death of many tumor cells. The effects of antivascular drugs are frequently short term, with regrowth beginning less than 24 hours posttreatment. This study investigated the duration of the response to the vascular targeting agent, ZD6126, of the GH3 prolactinoma, in which efficacy and dose-response have previously been demonstrated. GH3 prolactinomas were grown in the flanks of eight Wistar Furth rats. All animals were treated with 50 mg/kg ZD6126. The tumors were examined with dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) 24 hours pretreatment and posttreatment, and at a single time between 48 and 96 hours posttreatment. No evidence of recovery of perfusion was observed even at the longest (96-hour) time point. Involvement of a statistician at the project planning stage and the use of DCE-MRI, which permits noninvasive quantitation of parameters related to blood flow in intact animals, allowed this highly significant result to be obtained using only eight rats.

Tumour dose response to the antivascular agent ZD6126 assessed by magnetic resonance imaging.

ZD6126 is a vascular targeting agent that disrupts the tubulin cytoskeleton of proliferating neo-endothelial cells. This leads to the selective destruction and congestion of tumour blood vessels in experimental tumours, resulting in extensive haemorrhagic necrosis. In this study, the dose-dependent activity of ZD6126 in rat GH3 prolactinomas and murine RIF-1 fibrosarcomas was assessed using two magnetic resonance imaging (MRI) methods. Dynamic contrast-enhanced (DCE) MRI, quantified by an initial area under the time–concentration product curve (IAUC) method, gives values related to tumour perfusion and vascular permeability. Multigradient recalled echo MRI measures the transverse relaxation rate T2*, which is sensitive to tissue (deoxyhaemoglobin). Tumour IAUC and R2* (=1/T2*) decreased post-treatment with ZD6126 in a dose-dependent manner. In the rat model, lower doses of ZD6126 reduced the IAUC close to zero within restricted areas of the tumour, typically in the centre, while the highest dose reduced the IAUC to zero over the majority of the tumour. A decrease in both MRI end points was associated with the induction of massive central tumour necrosis measured histologically, which increased in a dose-dependent manner. Magnetic resonance imaging may be of value in evaluation of the acute clinical effects of ZD6126 in solid tumours. In particular, measurement of IAUC by DCE MRI should provide an unambiguous measure of biological activity of antivascular therapies for clinical trial.

Tumor vascular architecture and function evaluated by non-invasive susceptibility MRI methods and immunohistochemistry.

To investigate the physiological origins responsible for the varying blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) responses to carbogen (95% O(2)/5% CO(2)) breathing observed with different tumor types. Susceptibility contrast-enhanced MRI using the exogenous blood pool contrast agent NC100150 to determine blood volume and vessel size, and immunohistochemical-derived morphometric parameters, were determined in GH3 prolactinomas and RIF-1 fibrosarcomas, both grown in mice, which exhibited very different BOLD responses to carbogen. Administration of NC100150 increased the R(2)* and R(2) rates of both tumor types, and indicated a significant four-fold larger blood volume in the GH3 tumor. The ratio deltaR(2)*/deltaR(2) showed that the capillaries in the GH3 were two-fold larger than those in the RIF-1, in agreement with morphometric analysis. Carbogen breathing induced a significant 25% decrease in R(2)* in the GH3 prolactinoma, whereas the response in the RIF-1 fibrosarcoma was negligible. Low blood volume and small vessel size (and hence reduced hematocrit) are two reasons for the lack of R(2)* change in the RIF-1 with carbogen breathing. BOLD MRI is sensitive to erythrocyte-perfused vessels, whereas exogenous contrast agents interrogate the total perfused vascular volume. BOLD MRI, coupled with a carbogen challenge, provides information on functional, hemodynamic tumor vasculature.

Enhanced Uptake of Ifosfamide into GH3 Prolactinomas with Hypercapnic Hyperoxic Gases Monitored In Vivo by 31P MRS

Previously, 31P magnetic resonance spectroscopy (MRS) has been used to detect ifosfamide (IF) in vivo and to show that breathing carbogen (5% CO2/95% O2) enhances the uptake and increases the efficacy of IF in rat GH3 prolactinomas [Rodrigues LM, Maxwell RJ, McSheehy PMJ, Pinkerton CR, Robinson SP, Stubbs M, and Griffiths JR (1997). In vivo detection of ifosfamide by 31P MRS in rat tumours; increased uptake and cytotoxicity induced by carbogen breathing in GH3 prolactinomas. Br J Cancer 75, 62-68]. We now show that other hypercapnic and/or hyperoxic (5% CO2 in air, 2.5% CO2 in O2) gas mixtures also increase the uptake of IF into tumors, measured by 31P MRS. All gases caused an increased uptake (Cmax) of IF compared to air breathing, with carbogen inducing the largest increase (85% (P<.02) compared to 46% with 2.5% CO2 in O2 (P<.004) and 48% with 5% CO2 in air (P<.004)). The Tmax (time of maximum concentration in tumor posintravenous injection of IF) was significantly (P<.04) later in the cohort that breathed 5% CO2 in air. The increased uptake of IF with carbogen breathing was selective to tumor tissue and there were no significant increases in any of the normal tissues studied, suggesting that any host tissue toxicity would be minimal. Carbogen breathing by patients causes breathlessness. There was no significant difference in IF uptake between breathing carbogen and 2.5% CO2 in O2 and, therefore, the ability of 2.5% CO2 in O2 to also increase IF uptake may be clinically useful as it causes less patient discomfort.

In vivo hyperpolarized 129Xe NMR spectroscopy in tumors.

The first in vivo hyperpolarized 129Xe NMR study in experimental tumors is presented. Hyperpolarized 129Xe was dissolved in solutions, and was injected intratumorally in GH-3 prolactinomas in rats and RIF-1 fibrosarcomas in mice. The 129Xe NMR spectra and apparent spin-lattice relaxation times in the two tumor types present characteristic differences. These differences are discussed in terms of xenon exchange between the carrier medium and the tissue compartments.

Effects of different levels of hypercapnic hyperoxia on tumour R2∗ and arterial blood gases

The hypercapnia induced by carbogen (95% O2/5% CO2) breathing, which is being re-evaluated as a clinical radiosensitiser, causes patient discomfort and hence poor compliance. Recent preclinical and clinical studies have indicated that the CO2 content might be lowered without compromising increased tumour oxygenation and radiosensitisation. This preclinical study was designed to see if lower levels of hypercapnia could evoke similar decreases in the transverse relaxation rate R2∗ of rodent tumours to those seen with carbogen breathing. The response of rat GH3 prolactinomas to 1%, 212% and 5% CO2 in oxygen, and 100% O2 breathing, was monitored by non-invasive multi-gradient echo MRI to quantify R2∗. As the oxygenation of haemoglobin is proportional to the blood paO2 and therefore in equilibrium with tissue pO2, R2∗ is a sensitive indicator of tissue oxygenation. Hyperoxia alone decreased R2∗ by 13%, whilst all three hypercapnic hyperoxic gases decreased R2∗ by 29%. Breathing 1% CO2 in oxygen evoked the same decrease in R2∗ as carbogen. The ΔR2∗ response is primarily consistent with an increase in blood oxygenation, though localised increases in tumour blood flow were also identified in response to hypercapnia. The data support the concept that levels of hypercapnia can be reduced without loss of enhanced oxygenation and hence potential radiotherapeutic benefit.

Effects of nicotinamide and carbogen on tumour oxygenation, blood flow, energetics and blood glucose levels.

Both host carbogen (95% oxygen/5% carbon dioxide) breathing and nicotinamide administration enhance tumour radiotherapeutic response and are being re-evaluated in the clinic. Non-invasive magnetic resonance imaging (MRI) and 31P magnetic resonance spectroscopy (MRS) methods have been used to give information on the effects of nicotinamide alone and in combination with host carbogen breathing on transplanted rat GH3 prolactinomas. Gradient recalled echo (GRE) MRI, sensitive to blood oxygenation changes, and spin echo (SE) MRI, sensitive to perfusion/flow, showed large signal intensity increases with carbogen breathing. Nicotinamide, thought to act by suppressing the transient closure of small blood vessels that cause intermittent tumour hypoxia, induced a small increase in blood oxygenation but no detectable change in perfusion/flow. Carbogen combined with nicotinamide was no more effective than carbogen alone. Both carbogen and nicotinamide caused significant increases in the nucleoside triphosphate/inorganic phosphate (βNTP/P i) ratio, implying that the tumour cells normally receive sub-optimal substrate supply, and is consistent with either increased glycolysis and/or a switch to more oxidative metabolism. The most striking observation was the marked increase in blood glucose (twofold) induced by both nicotinamide and carbogen. Whether this may play a role in tumour radiosensitivity has yet to be determined. Copyright 2000 Cancer Research Campaign© 2000 Cancer Research Campaign

Formula omitted]w and [formula omitted]xygenation [formula omitted]ependent (FLOOD) contrast MR imaging to monitor the response of rat tumors to carbogen breathing

Gradient recalled echo (GRE) images are sensitive to both paramagnetic deoxyhaemoglobin concentration (via T ∗ 2) and flow (via T ∗ 1). Large GRE signal intensity increases have been observed in subcutaneous tumors during carbogen (5% carbon dioxide, 95% oxygen) breathing. We term this combined effect flow and oxygenation-dependent (FLOOD) contrast. We have now used both spin echo (SE) and GRE images to evaluate how changes in relaxation times and flow contribute to image intensity contrast changes. T 1-weighted images, with and without outer slice suppression, and calculated T 2, T ∗ 2 and “flow” maps, were obtained for subcutaneous GH3 prolactinomas in rats during air and carbogen breathing. T 1-weighted images showed bright features that increased in size, intensity and number with carbogen breathing. H&E stained histological sections confirmed them to be large blood vessels. Apparent T 1 and T 2 images were fairly homogeneous with average relaxation times of 850 ms and 37 ms, respectively, during air breathing, with increases of 2% for T 1 and 11% for T 2 during carbogen breathing. The apparent T ∗ 2 over all tumors was very heterogeneous, with values between 9 and 23 ms and localized increases of up to 75% during carbogen breathing. Synthesised “flow” maps also showed heterogeneity, and regions of maximum increase in flow did not always coincide with maximum increases in T ∗ 2. Carbogen breathing caused a threefold increase in arterial rat blood p aO 2, and typically a 50% increase in tumor blood volume as measured by 51Cr-labelled RBC uptake. The T ∗ 2 increase is therefore due to a decrease in blood deoxyhaemoglobin concentration with the magnitude of the FLOOD response being determined by the vascular density and responsiveness to blood flow modifiers. FLOOD contrast may therefore be of value in assessing the magnitude and heterogeneity of response of individual tumors to blood flow modifiers for both chemotherapy, anti-angiogenesis therapy in particular, and radiotherapy.

Causes and consequences of acidic pH in tumors: a magnetic resonance study.

A consequence of metabolism in any tissue is the formation of hydrogen ions, which are actively transported out of the cell. However, although most solid tumors maintain their intracellular pH (pHi) within a narrow range to provide a favorable environment for various intracellular activities, their extracellular pH (pHe) is on average about 0.2 pH units more acid. It is important to understand the relationship between tumor metabolism and pH, and how it differs from that of normal tissue and to ask the question: How does an understanding of pH and tumor metabolism affect strategies for therapeutic approaches? Although, in vitro, isolated cell experiments have shown positive correlations between pHi and pHe the relationship is complex and somewhat dependent on experimental conditions. Measurement of pHi in solid tumors by non-invasive Magnetic Resonance Spectroscopy (MRS) has been possible for some time now and recently several specific markers for measuring pHe have become available. As a result we have been able to study the relationship between pHi and pHe in vivo in several different solid tumor types. In only one tumor type (HT29 xenografts) was there a significant correlation between pHi and pHe; in 3 other tumor types (RIF-1 in mice, GH3 prolactinomas and H9618a in rats) there was no correlation. A significant correlation between pHi and NTP/Pi ratios was seen across all tumor types. Theoretical considerations of causes of tumor acidity, hypotheses to explain extracellular acidity and the possibility that low pHe might be an intrinsic feature of the tumor phenotype and not merely the consequence of metabolic activity have been discussed. In addition the consquences for concepts of treatment based on pH are considered.

Tumour response to hypercapnia and hyperoxia monitored by FLOOD magnetic resonance imaging.

Flow and oxygenation dependent (FLOOD) MR images of GH3 prolactinomas display large intensity increases in response to carbogen (5% CO2/95% O2) breathing. To assess the relative contributions of carbon dioxide and oxygen to this response and the tumour oxygenation state, the response of GH3 prolactinomas to 5% CO2/95% air, carbogen and 100% O2 was monitored by FLOOD MRI and PO2 histography. A 10-30% image intensity increase was observed during 5% CO2/95% air breathing, consistent with an increase in tumour blood flow, as a result of CO2-induced vasodilation, reducing the concentration of deoxyhaemoglobin in the blood. Carbogen caused a further 40-50% signal enhancement, suggesting an additional improvement due to increase blood oxygenation. A small 5-10% increase was observed in response to 100% O2, highlighting the dominance of CO2-induced vasodilation in the carbogen response. Despite the large FLOOD response, non-significant increases in tumour pO2 were observed in response to the three gases. Tissue pO2 is determined by the balance of oxygen supply and demand, hence increased blood flow/oxygenation may not necessarily produce a large increase in tissue PO2. The FLOOD response is determined by the level of deoxygenation of blood, the size of this response relating to vascular density and the potential of high-oxygen content gases to improve the oxygen supply to tumour tissue.