Loreta M. Rodrigues

Magnetic resonance imaging techniques for monitoring changes in tumor oxygenation and blood flow

The application of functional magnetic resonance (MR) imaging techniques to the measurement of oxygenation and blood flow in tumors is described. Gradient recalled echo MR imaging (GRE-MRI) offers a real-time noninvasive method for monitoring tumor response to vasomodulators such as carbogen (95% O2/5% CO2) breathing in attempts to overcome tumor hypoxia and improve treatment efficacy. Although the response is tumor-type dependent, increases in signal intensity of up to 100% have been observed in several animal tumor types. Responses are also seen in human tumors. The observed increases in GRE-MRI signal intensity are due to a combination of a reduction of deoxyhemoglobin in the blood causing changes in the MR imaging relaxation time T2* and changes in blood flow and may also reflect the capillary density. Thus, the magnitude of the GRE image intensity change gives an indication of the potential response of an individual tumor to treatments that aim to improve tissue oxygenation and therefore how the tumor may respond to therapy. In addition, carbogen breathing by the host has been shown to increase the uptake and efficacy of chemotherapeutic agents in animal tumors.

The response to carbogen breathing in experimental tumour models monitored by gradient-recalled echo magnetic resonance imaging

Gradient-recalled echo magnetic resonance imaging (GRE MRI), which gives information on blood flow and oxygenation changes (Robinson SP, Howe FA, Griffiths JR 1995, Int J Radiat Oncol Biol Phys 33: 855), was used to observe the responses of six rodent tumour models to carbogen breathing. In one transplanted rat tumour, the Morris hepatoma 9618a, and a chemically induced rat tumour, the MNU-induced mammary adenocarcinoma, there were marked image intensity increases, similar to those previously observed in the rat GH3 prolactinoma. In contrast, the rat Walker carcinosarcoma showed no response. In two mouse tumours, the RIF-1 fibrosarcoma and the human xenograft HT29, carbogen breathing induced a transient fall in signal intensity that reversed spontaneously within a few minutes. The rat GH3 prolactinoma was xenografted into nude mice, and an increase in image intensity was found in response to carbogen, suggesting that any effects that carbogen may have had on the host were not significant determinants of the tumour response. The increases in GRE image intensity of the MNU, H9618a and GH3 tumours during carbogen breathing are consistent with increases in tumour oxygenation and blood flow, whereas the responses of the RIF-1 and HT29 tumours may be the result of a transient steal effect followed by homeostatic correction.

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. Copyright

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.

The effect of blood flow modification on intra- and extracellular pH measured by 31P magnetic resonance spectroscopy in murine tumours.

Intra- and extracellular pH (pHi and pHe) were measured simultaneously by 31P magnetic resonance spectroscopy (MRS) in CaNT tumours before and after blood flow modification. Before modification, pHi was 7.1 +/- 0.09 (n = 11) and pHe [measured with an MRS-visible extracellular marker, 3-aminopropyl phosphonate (3-APP)] was 6.7 +/- 0.05 (n = 8). Chemical shift imaging and localised MRS experiments showed that the 3-APP signal was only from the tumour, not surrounding tissue. After modification by vascular occlusion, independent of whether tumours were maintained at room temperature (22-24 degrees C) or kept warm (33-35 degrees C), there was a decrease in pHi and pHe with pHi decreasing to a greater extent. Qualitatively similar results were found using flavone acetic acid (FAA) as a blood flow modifier; only four out of nine tumours responded to FAA. Concomitant with the reduction of the pH gradient after modification was a decrease in the phosphorylation state of the adenine nucleotides measured either as ATP/Pi by MRS or [ATP]/[ADP][P(i)] in tumour extracts. These results indicate that the intracellular uptake of chemotherapeutic drugs which are dependent on the transmembrane pH gradient will not be enhanced in cells made ischaemic as a result of vascular shutdown.

Protection of cells against membrane damage by haemolytic agents: divalent cations and protons act at the extracellular side of the plasma membrane

The protective effect of Ca2+, Zn2+ and H+ against membrane damage induced by different haemolytic agents has been studied by measuring monovalent cation leakage and haemolysis of erythrocytes, and phosphoryl[3H]choline and adenine nucleotide leakage from Lettre cells prelabelled with [3H]choline. The protective effect of Ca2+ and Zn2+ on erythrocytes damaged by Staphylococcus aureus alpha-toxin, Sendai virus or melittin is unaffected by the addition of A23187, even though this ionophore greatly increases the uptake of 45Ca2+ or 65Zn2+. The same result has been found for the protective effect of Zn2+ on Lettre cells damaged by S. aureus alpha-toxin, Sendai virus, melittin or Triton X-100. Leakage of phosphoryl[3H]choline from prelabelled Lettre cells is inhibited if extracellular pH is lowered; lowering the intracellular pH without affecting the extracellular pH, affords little protection. It is concluded that Ca2+, Zn2+ and H+ protect cells against membrane damage induced by haemolytic agents by an action at the extracellular side of the plasma membrane.

Assessment of induced rat mammary tumour response to chemotherapy using the apparent diffusion coefficient of tissue water as determined by diffusion-weighted 1H-NMR spectroscopy in vivo

Chemosensitivity of N-methyl-N-nitrosourea-induced rat mammary tumours treated with 5-fluorouracil at a dose of 100 mg kg(-1) i.p. was assessed by using diffusion-weighted 1H-MRS to measure the average diffusion coefficient (ADC) of water in the tumour tissue. ADC measurements prior to any therapy correlated positively with necrotic fraction. Tumours with low initial ADC ( 1.2 x 10(9) m2 s(-1)) showed a decrease. All tumours decreased significantly in volume (P < 0.05) 2, 5 and 7 days after treatment. At day 7 post-treatment, tumours with a high pre-treatment ADC started to regrow. The initial ADC value, as well as changes after treatment predict tumour chemosensitivity, which could be clinically relevant.

In vivo detection of ifosfamide by 31P-MRS in rat tumours: increased uptake and cytotoxicity induced by carbogen breathing in GH3 prolactinomas.

The direct detection and monitoring of anti-cancer drugs in vivo by magnetic resonance spectroscopy (MRS) may lead to improved anti-cancer strategies. 31P-MRS has been used to detect and quantify ifosfamide (IF) in vivo in GH3 prolactinomas and N-methyl-N-nitrosourea (MNU)-induced mammary tumours in rats. The average concentration of IF in the GH3 prolactinoma over the first 2 h following a dose of 250 mg kg-1 i.v. was calculated to be 0.42 micromol g-1 wet weight, with a half-life of elimination (t1/2) of 2-4 h. Carbogen (95% oxygen/5% carbon dioxide) breathing increased the amount of IF taken up by the GH3 prolactinoma by 50% (P<0.01) to 0.68 micromol g-1 wet weight, although t1/2 elimination rates were unchanged. IF was also detected in the liver in vivo, with a t1/2 of about 1 h. Carbogen breathing did not affect the maximum peak area (Cmax) or the t1/2 in the liver. Most importantly, the carbogen-induced increase in IF uptake by the tumour caused significant growth delay at all time points in the GH3 tumour growth between day 5 and day 12 (P< 0.01) compared with IF alone. These findings show that carbogen breathing has potential for increasing the efficacy of anti-cancer drugs. Isolated GH3 cells were sensitive to the parent drug (IF) in vitro (IC50 = 1.3 +/- 0.2 mM) suggesting that the GH3 cells may be either expressing P450 enzymes or are sensitive to the parent drug per se.

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

The hypercapnia induced by carbogen (95% O(2)/5% CO(2)) 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 CO(2) 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 R(2)* of rodent tumours to those seen with carbogen breathing. The response of rat GH3 prolactinomas to 1%, 212% and 5% CO(2) in oxygen, and 100% O(2) breathing, was monitored by non-invasive multi-gradient echo MRI to quantify R(2)*. As the oxygenation of haemoglobin is proportional to the blood p(a)O(2) and therefore in equilibrium with tissue pO(2), R(2)* is a sensitive indicator of tissue oxygenation. Hyperoxia alone decreased R(2)* by 13%, whilst all three hypercapnic hyperoxic gases decreased R(2)* by 29%. Breathing 1% CO(2) in oxygen evoked the same decrease in R(2)* as carbogen. The DeltaR(2)* 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.

A pharmacokinetic and pharmacodynamic study In vivo of human HT29 tumours using 19F and 31P magnetic resonance spectroscopy

19F-MRS (magnetic resonance spectroscopy) was used to study the pharmacokinetics of 5-fluorouracil (5-FU) in human (HT29) tumour xenografts, with and without pretreatment of the mice using either thymidine (40 min) or interferon-alpha (2 and 24 h). A 200 mg/kg i.p. bolus dose of 5-FU was eliminated from control tumours with a t1/2 of 25.4 +/- 2 min (mean +/- SEM, n = 11), while both thymidine (500 mg/kg) and interferon (50,000 IU/mouse) significantly increased t1/2 to 36.5 +/- 6.1 (n = 5) and 48.1 +/- 13.6 min (n = 4), respectively (P = 0.04, Gabriels ANOVA). Thymidine increased 5-FU anabolism to cytotoxic 5-fluoronucleotides, and decreased the amount of tumour catabolites; the latter probably recirculated from liver since isolated HT29 cells did not catabolize 5-FU. These in vivo observations were confirmed by 19F-MRS quantification of tumour extracts. Interferon did not significantly affect 5-FU metabolism in the tumour or liver, nor the 5-FU t1/2 in liver. Treatment of tumours with 5-FU or interferon had no effect on tumour growth, whereas the combination strongly inhibited growth. 31P-MRS of HT29 tumours showed that 2 and 24 h after i.p. injections of interferon there was a significant increase in the pHint of 0.3 +/- 0.04 units (P = 0.002), while pHext and the tumour NTP/Pi ratio were unchanged. The large increase in the negative pH gradient (-delta pH) across the tumour plasma membrane caused by interferon suggest the delta pH may be a factor in tumour retention of 5-FU, as recently shown in isolated tumour cells.