Multimodality imaging of hypoxia in preclinical settings

Abstract

Hypoxia has long been recognized to influence solid tumor response to therapy. Increasingly, hypoxia has also been implicated in tumor aggressiveness, including growth, development and metastatic potential. Thus, there is a fundamental, as well as a clinical interest, in assessing in situ tumor hypoxia. This review will examine diverse approaches focusing on the pre-clinical setting, particularly, in rodents. The strategies are inevitably a compromise in terms of sensitivity, precision, temporal and spatial resolution, as well as cost, feasibility, ease and robustness of implementation. We will review capabilities of multiple modalities and examine what makes them particularly suitable for investigating specific aspects of tumor pathophysiology. Current approaches range from nuclear imaging to magnetic resonance and optical, with varying degrees of invasiveness and ability to examine spatial heterogeneity, as well as dynamic response to interventions. Ideally, measurements would be non-invasive, exploiting endogenous reporters to reveal quantitatively local oxygen tension dynamics. A primary focus of this review is magnetic resonance imaging (MRI) based techniques, such as ¹⁹F MRI oximetry, which reveals not only hypoxia in vivo, but more significantly, spatial distribution of pO₂ quantitatively, with a precision relevant to radiobiology. It should be noted that pre-clinical methods may have very different criteria for acceptance, as compared with potential investigations for prognostic radiology or predictive biomarkers suitable for use in patients.

1. Introduction

Hypoxia and tissue oxygenation comprise a vast field of study relevant to diverse diseases. This review focuses on its relevance to solid in situ tumor development, progression, and primarily response to therapy. Studies range from molecular biology to therapeutic oncology with a common thread of imaging based on developments in reporter molecules, physics, engineering and data processing.

Ever since the classic studies of Gray et al. fifty years ago (1), it has been appreciated that hypoxia can influence the efficacy of radiotherapy on tumors. There have been many attempts to modulate tumor hypoxia in order to enhance radiotherapy, but translation to the clinic has shown marginal efficacy (2). The meta analysis by Overgaard et al. (3) of some 10,000 patients indicated a clinical benefit for manipulating tumor hypoxia. However, the overall conclusion was that there was a pressing need to identify those tumors (viz. patients), who would actually benefit.

In terms of hypoxia in human tumors, we perceive three needs: 1) to develop an effective method of detecting hypoxia non-invasively in tumors of patients prior to therapy; 2) to assess the ability to modulate tumor hypoxia to overcome resistance to therapy; 3) to exploit hypoxia using specific individualized patient therapies, which are effective on hypoxic cells. We believe we are at a historic juncture, where we not only have technologies for identifying hypoxia, but more importantly methods of tailoring therapy successfully to accommodate or exploit the killing of hypoxic cells.

One aspect of pre-clinical research is simply to develop methods satisfying the clinical requirements. However, for pre-clinical applications themselves, we perceive slightly modified needs, firstly: effective methods of assaying tumor hypoxia, and secondly: well-characterized animal models with tumors exhibiting well-defined differential levels of hypoxia and responses to interventions. Criteria for useful methods are also somewhat different, e.g., imaging methods may rely on reporter molecules, which are efficient and ethical for use in small animal studies, but do not have, and might not meet, requirements for patients (notably, IND). The requirements are not necessarily less stringent.

Early work on effects of hypoxia examined cells in vitro, where ambient oxygen concentrations are readily controlled. In vivo, hypoxia may be achieved by clamping the blood supply to a tumor (4), but other levels of oxygenation reflect the interplay of supply and consumption (5). Given the importance of hypoxia many methods have been developed for probing tumor oxygenation including hypoxia reporter agents, polarographic electrodes, fiber optic probes, NIR spectroscopy, and various NMR techniques, but most are either highly invasive, lack spatial resolution or lack the ability to dynamically and quantitatively sample the response to intervention (2, 6–8) (Table 1).

Table 1.

Tumor Oximetry methods

Technique	Reporter	Parameter	Characteristics	References
Magnetic resonance
BOLD	Magnetic fields created by deoxyHb Vascular water	R2*; ΔR2*ΔSI (T2*w)	Imaging at high spatial and temporal resolution. Non- invasive. Endogenous contrast with hyperoxic gas challenge. Potential interference from blood flow, volume.	(92, 96, 100, 102, 127)
TOLD	Magnetic fields created by O2 Tissue water	R1; ΔR1ΔSI (T1w)	Endogenous contrast or hyperoxic gas challenge.	(109, 111, 128)
19F MR, e.g., FREDOM	HFB	R1	Map multiple locations each 8 mm3, 6.5 mins (recently 90 s), direct injection of reporter into tissue using 32G needle	(6, 82, 89, 100, 117)
19F MRI	PFC emulsion	R1, R2	IV, may bias measurements to perfused regions	(76, 77, 129, 130)
PISTOL	HMDSO	R1	Map multiple locations in 4.5 mins for pO2 map- procedure analogous to FREDOM	(91)
DCE MRI	Gd-DTPA	Contrast kinetics	IV, Maps, mins -parameters have been related to pO2	(131–133)
19F NMR	F-nitroimidazoles	Signal intensity	Hypoxia, not pO2	(38, 39, 125)
ESR/EPR	Chars; Phthalocyanine Nitroxides	Linewidth	Needle 23 G IT, single location or maps. Problem of reporter decay	(64, 65, 69)
Optical
Phosphorescence	Pd-complex	Lifetime	Vascular signal limited by tissue light penetration	(134)
NIR	Oxy and deoxy hemoglobin	Relative absorption	Vascular signal	(68, 82, 135)
FLI and BLI with reporter genes under HRE control	eGFP, Luciferase	Signal intensity	Limited tissue light penetration BLI requires luciferin substrate	(19)
Nuclear
PET	18F misonidazole labeled with 18F 18F EF5 Cu-ATSM (60,62,64, 67Cu)	Tumor to muscle ratio	Non invasive. High sensitivity. Only viable hypoxic cells give signal; radioactive compounds. short half life; small range of pO2 values covered; low spatial resolution. Cu-ATSM uptake perturbed by MDR transporters.	(17, 34, 46, 47)

2.0 Hypoxia

The concept of hypoxia may be debated, but presumably indicates oxygen insufficiency. Anoxia is presumed to be complete absence of molecular oxygen and this may be achieved in model systems and cell cultures. Indeed, cell culture incubators are available to exclude and eliminate oxygen, as required for the culture of facultative anaerobes. More commonly, cell cultures are saturated with a gaseous atmosphere of specific oxygen concentration and cells are adapted to growth under low pO₂ saturation.

In the heart and brain, hypoxia may be attributed to ischemia, where the delivery of oxygen is insufficient to maintain normal function. Ischemia may be total, or partial, and it may manifest itself as chest pain, angina or even be silent for many years, while chronic damage is established. Hypoxia may occur due to local lack of vascular flow or insufficient hemoglobin saturation. An acute myocardial infarct will result in rapid oxygen depletion and loss of mechanical function. We have shown, as expected, using ¹⁹F NMR of perfluorocarbons sequestered in the myocardium that hypoxiation is much more rapid in a beating heart than an arrested heart, and indeed, the rate of loss of developed pressure is directly proportional to decrease in pO₂ (9).

However, in tumors, a definition of hypoxia may be far more complex, since the tissue is aberrant with no particular normal function, yet well documented ability to adapt to stress. Hypoxia has been shown to stimulate mutations, and hypoxic stress appears to stimulate angiogenesis and metastasis, particularly, as a result of intermittent bouts of hypoxic stress (10–12). Tumor cells may survive in quite hypoxic conditions, but for a solid tumor to grow beyond a few hundred microns in diameter, an angiogenic switch is required to generate new blood vessels for the delivery of nutrients and oxygen (13). In hypoxic conditions, tumor cells release angiogenic factors stimulating blood vessel sprouting and a new vascular network. Figure 1 shows typical chaotic vascular extent, and thus tumors are expected to be hypoxic. In terms of Figure 1d, it is noteworthy that externalization of phosphatidylserine (PS) to the luminal surface of the vascular endothelium is stimulated by stress including hypoxia (14). Consequently, phosphatidylserine is a marker of tumor vasculature and a potential target for anti-cancer drugs. The exposed PS is one of the most specific markers of tumor vasculature yet discovered and provides an excellent marker for imaging with radionuclide-bavituximab conjugates (15).

Figure 1. Tumor vascular heterogeneity and hypoxia.

Panels each provide evidence for vascular heterogeneity based on in vivo and ex vivo imaging:

A) Mammary adenocarcinoma 13762NF tumors were grown using a kidney tissue-isolated preparation in adult female Fischer 344 rats. Perfusion of the abdominal aorta proximal to the renal artery with Batson’s #17 polymer provided an ex vivo 3D representation by means of corrosion casting. Vascular casts were sputtered with gold-palladium prior to Scanning Electron Microscopy. Disorganized vasculature is apparent with many tortuous microvessels and blind ends (data obtained in collaboration with Andrew Abbott and Dr. C. Gilpin).

B) Immunohistochemistry from similar 13762NF tumor. Pimonidazole was infused and 60 mins later Hoechst 33342 dye (seen in blue) was infused IV with tumor excision 60 s later. Frozen 6 μm-thick sections were stained using anti-CD31 mAb (red). Extravasated Hoechst dye surrounded the red blood vessels. Scale bar 100 μm, original magnification x100.

C) Separate section of the same tumor as B. Perfusion is shown by Hoechst dye (blue) and hypoxia in green.

D) Image slice from 3D PET of 13762NF tumor bearing rat 24 hrs after infusion of 41 μCi ⁷⁴As-SATA-labeled bavituximab - a humanized monoclonal antibody targeting exposed phosphatidylserine of the outer leaflet of stressed vasculature. The tumor (arrow) shows distinct vascular heterogeneity. At this stage, much of the activity was likely still circulating in the vasculature.

E) ¹H MRI of slice of Dunning prostate R3327-AT1 tumor obtained 20 mins after IV infusion of Gd-DTPA contrast agent (Magnevist).

F) Same as E with selected regions of interest

G) Time course of signal intensity variations (contrast) in tumor regions delineated in F, showing distinct heterogeneity of perfusion (data obtained in collaboration with Dr. P. Antich).

H) Autoradiography of slice of excised AT1 tumor following infusion of the vascular volume marker ¹²⁵I-labeled albumin, revealing similar heterogeneity to that detect by MRI in vivo in E-G (data obtained in collaboration with Dr. J Anderson).

The expression of many proteins is influenced by hypoxia (16, 17) and hypoxia sensitive promoter regions have been developed to generate hypoxia responsive reporter genes. A specific promoter regulating a reporter gene, such as luciferase or fluorescent protein (e.g., eGFP), can selectively turn on expression under hypoxic conditions. Often, multiple promoter copies are exploited to avoid expression leakage and an oxygen degradation domain may be incorporated (18). This approach requires transfection of cells and reporter proteins may remain for several hours after amelioration of hypoxia, preventing acute dynamic studies. This approach is particularly suitable for observing onset of chronic hypoxia (19). In small animals, optical imaging has the advantages of being cheap, generally simple to implement on commercial instruments, and allows high throughput. Fluorescent proteins may be detected non-invasively, although depth of light penetration limits studies to small animals or surface tissues. Luciferase does require administration of luciferin substrate.

It has long been appreciated that hypoxic tumor cells are more resistant to radiotherapy (1). Indeed, a threefold increase in radio-resistance may occur when cells are irradiated under hypoxic conditions compared with pO₂ > 15 torr for a given single radiation dose. However, recent modeling has indicated that the proportion of cells in the range 0 – 20 torr may be most significant in terms of surviving a course of fractionated radiotherapy (20). The clinical significance of hypoxia for radiotherapy was recently illustrated by modeling the radiation response of a heterogeneous cell population. Based on an oxygen enhancement ratio of 3.0, Fowler et al. (21) demonstrated that while a fractionated dose of 70–80 Gy would lead to reduction in cell survival from 1 to 10⁻¹² for a well-oxygenated tumor, a similar reduction in survival would require a dose exceeding 200 Gy for a tumor containing 20% hypoxic cells. In the clinical setting, we expect hypoxia to be even more important for high dose fractionation protocols, which are gaining feasibility based on precise treatment plans and effective targeting. Timmerman et al. (22) recently reported results of The Radiation Therapy Oncology Group (RTOG) protocol RTOG 0236 Phase II trial utilizing stereotactic body radiation therapy (SBRT) with ablative prescription dose to treat early-stage medically inoperable non–small-cell lung cancer (NSCLC) patients. Using three doses of 20 Gy in less than two weeks, the estimated 2-year survival exceeded 90%, which is a remarkable improvement over traditional therapy. When few doses are applied, the hypoxic fraction is expected to have much greater impact on radioresistance, since there is no opportunity for the progressive reoxygenation and repopulation attributed to long-term fractionated irradiation and thus methods of assessing hypoxia are increasingly relevant.

When hypoxia is identified several options arise: i) “dose-painting”, booster regimens and IMRT have been proposed and are being tested (23–26), ii) administering adjuvant hypoxic cell selective cytotoxins: clinical trials with tirapazamine were halted, but new, potentially, more effective drugs are being developed, e.g., AQ4N and TH302 (27, 28); iii) a simpler intervention would be breathing hyperoxic gas to ameliorate the hypoxia. Indeed, many clinical trials have explored oxygen, carbogen or hyperbaric gas breathing, but with limited success, likely due to the inability to identify those patients with hypoxic tumors, who would benefit. By analogy, assaying HER2/neu status is vital to the effective use of Herceptin (Trastuzumab) to treat breast cancer patients. Thus, a crucial concern is identifying those patients, who may need the adjuvant intervention and benefit and there is an urgent need to develop methods of indentifying potentially resistant hypoxic tumors and implementing adjuvant interventions. We note the report from Nijmegen (29), which showed that patients with large head and neck hypoxic tumors (identified using pimonidazole in biopsies) could be brought into the realm of effective response by applying the ARCON protocol (Accelerated Radiation therapy with Carbogen and Nicotinamide).

2.1 Radionuclide imaging of hypoxia: PET, SPECT and γ-scintigraphy

Given the importance of hypoxia diverse classes of reporter molecules have been developed to reveal hypoxia, e.g., pimonidazole (29, 30), EF5 (31, 32), CCI-103F (33), Cu-ATSM (34), galactopyranoside IAZA (35). Many reporters are based on the structure of misonidazole, which had been explored extensively as a radiation sensitizer. Unacceptable toxicity (neuropathy) halted clinical applications, but it was realized that reporter molecules could be used at much lower concentrations, e.g., 15 μg as opposed to 10 g per patient (17) allowing effective application. Indeed, beyond pre-clinical applications EF5, FAZA, Cu-ATSM and F-MISO are being evaluated in ongoing clinical trials (http://clinicaltrials.gov/).

Following IV infusion, the agents diffuse into tissues, where they are reduced forming highly reactive intermediates. In the presence of oxygen, they are reoxidized and ultimately clear from the body. However, in the absence of molecular oxygen, further reductions occur yielding radical anions and reactive alkylating amine derivatives, which are trapped in the cell (17). In biopsies, EF5 and pimonidazole are both routinely used in conjunction with fluorescent immunohistochemical analysis providing microscopic indications of local hypoxia (e.g., Fig. 1c shows hypoxia in a rat breast tumor distant from the well perfused regions) (36). EF5, pimonidazole, and Cu-ATSM are currently being tested in clinical trials and correlations have been reported relating uptake to clinical outcome (29, 32, 37). Over the past 20 years, many variant reporter molecules have been proposed focusing on modifying lipophilicity, reduction potential and reactivity. MRI has been applied to several fluoronitroimidazole derivatives including EF5 and SR-4554 (38, 39).

Incorporation of radionuclides has facilitated non-invasive investigations using PET, SPECT or γ-scintigraphy, as reviewed extensively by others recently (8, 17, 40–42). The most widely used reporter has been fluoromisonidazole (F-MISO), which has been tested extensively in animals (Figure 2) and is being evaluated in clinical trials. F-18 has a relatively short half-life (110 mins), but an efficient synthesis was developed based on ¹⁸F-fluoride and a pure racemic mixture suitable for injection into patients can be produced in 35 minutes with a specific activity of 250 GBq/μmol (17). Thus, a typical patient dose of 260 MBq requires only 0.2 μg. Patients are normally imaged 1.5 hours after administration.

Figure 2. Comparison of F-MISO and 124-IAZAG in rat liver tumors by PET.

Images show various time points after the administration on consecutive days of ¹⁸F-FMISO (2 mCi; upper row) or ¹²⁴I-IAZG (1 mCi; lower row) to a rat with Morris hepatoma McA-R-7777. ¹⁸F-FMISO localized to the same tumor regions as ¹²⁴I-IAZG. The contrast ratios (tumor/background) reached similar values for the two hypoxia tracers, but at later times for ¹²⁴I-IAZG than for ¹⁸F-FMISO and, hence poorer count statistics so that the ¹⁸F-FMISO images were of superior diagnostic image quality to the ¹²⁴I-IAZG images. H=heart; L=liver; St=stomach; LT=liver tumor; PT=peritoneal tumor. Reprinted by permission of (44)

Early autographic studies based on tritiated F-MISO showed effective penetration into cultured cell spheroids with greatest concentration being at the interface of viable cells and the necrotic center. Under anoxic conditions, binding was found to be 25-fold greater than in normoxic conditions, where binding dropped to 40% at 4 mmHg (torr). In vivo, there is concern that distribution and uptake may be influenced by relative blood flow, which is likely to be particularly low in hypoxic regions. Correlative studies with the blood flow indicator ¹⁴C-iodoantipyrine showed no correlation between flow and F-MISO binding (17). Krohn et al. (2) state that a tumor to background ratio less than unity is an indication of normoxic tissues, while values greater than 1.2 infer hypoxia. However, slow washout is a concern and alternative fluorinated derivatives have been evaluated, particularly exploring differential lipophilicity based on degree of fluorination (Table 2) (17). FAZA showed much faster clearance from the blood of mice and while tumor uptake was less, the tumor to blood was significantly greater than for F-MISO (43).

Table 2.

Radionuclide approaches to assessing tumor hypoxia

			references
Agent	nuclide	Chemistry for synthesis	Pre-clinical applications	Clinical applications
F-MISO (18F-fluoromisonidazole)	18F	(136)	(43, 137)	(138)
EF1 (2-(2-Nitroimidazol-1H-yl)-N-(3-[18F]fluoropropyl)acetamide)	18F	(139)	(140)	none
EF3 (2-(2-nitroimidazol-1-yl)-N-(3[F-18], 3,3-trifluoropropyl) acetamide)	18F	(141)	(142)	(143)
EF5 (2(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3- pentafluoropropyl)acetamide)	18F	(144)	(145, 146)	(147)
FETA (fluoroetanidazole)	18F	(148)	(149)	none
FETNIM (Fluorine-18 fluoroerythronitroimidazole)	18F	(150)	(151)	(152)
FAZA (fluoroazomycin arabinoside)	18F	(153)	(43, 154)	none
BMS181321 and other 99mTc-labeled nitroimidazoles HL-91 (PROGNOX)	99mTc	(155)	(156) (157, 158)	(159)
IAZG (iodo-azomycin galactopyranoside)	123I SPECT 124I PET 125I SPECT 131I for therapy+ autoradiography	(160)	(161) (43, 44, 162, 163) (157) (164)	NCT00588276 http://clinicaltrials.gov/ct2/show/NCT00588276
Cu-ATSM (diacetyl-bis(N4- methylthiosemicarbazone))	60Cu PET 62Cu 64Cu PET	(165) (45)	(46, 47)	(34, 48) (166) (48)

Further information on many of these agents is available from the NIH MICAD data base http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=micad

Alternate agents use iodoazogalactosides or copper lactams and each is undergoing clinical trials (Table 2). I-124 allows PET and the longer half-life allows imaging after 24 to 48 hrs when background has cleared. Several studies have shown efficacy of this approach, but the signal to noise appears inferior to F-MISO (Fig. 2) (44). Iodine labeling has the added advantage of allowing several isotopes for different applications including PET (¹²⁴I), SPECT (^{123, 125}I), γ-scintigraphy (¹²⁵I) and radioimmunotherapy (¹³¹I), although instability and dehalogenation can be problematical. Copper based agents are attractive, since they also allow multiple isotopes with longer half lives (⁶⁰Cu t_½ = 23 mins, 93% β^{+; 61}Cu t_½ = 3.3 hrs, 61% β^{+; 62}Cu t_½ = 9.7 mins; ⁶⁴Cu t_½ = 12.7 hrs, 17.4% β⁺ for PET; and ⁶⁷Cu t_½ = 61 hrs for therapy) (45). Cu-ATSM shows specificity for hypoxic tissues and has shown correlation with clinical outcome for lung and cervical cancer patients (34, 37). However, uptake and retention appears to be inconsistent in some cells and may be influenced by protein expression related to multi drug resistance (46, 47). Early PET studies had used ⁶⁰Cu-PTSM, but recently Lewis et al. (48) showed that ⁶⁴Cu-PTSM was equally applicable in patients with cervical cancer and indeed the image quality was reported to be better (Figure 3). However, differences in image quality may be attributed to some variation in data acquisition including 2D versus 3D to avoid contamination from daughter nuclides. Copper-64 is increasingly available from commercial sources to generate such labels, e.g., MDSNordion (Canada), ACOM(Italy), Trace Life Sciences (United States), IBA Molecular (United States and Europe), and IsoTrace (United States). The longer half-life promises a more convenient radiopharmaceutical. Beyond nuclides of iodine and copper, one can envision an arsenic nitroimidazole, where the isotopes ^{72, 74 and 77}As provide diverse opportunities for PET and radiotherapy (15, 49).

Figure 3. Comparison of ⁶⁰Cu-ATSM and ⁶⁴Cu-ATSM in patients with cervical carcinoma using PET.

(A) Transaxial CT (top left) and ¹⁸F-FDG PET (top right) images of pelvis showing intense ¹⁸F-FDG uptake within known cervical tumor seen on CT. Transaxial 30- to 60-min summed images of ⁶⁰Cu-ATSM PET (bottom left) and ⁶⁴Cu-ATSM PET (bottom right) of pelvis at same level demonstrate mildly increased uptake within known primary cervical tumor.

(B) Transaxial coregistered ¹⁸F-FDG PET/CT (top left) and ¹⁸F-FDG PET (top right) images of pelvis show intense ¹⁸F-FDG uptake within cervical tumor. Transaxial 30- to 60-min summed images of ⁶⁰Cu-ATSM PET (bottom left) and ⁶⁴Cu-ATSM PET (bottom right) of pelvis at same level. Both patients showed similar patterns of ⁶⁰Cu-ATSM and ⁶⁴Cu-ATSM uptake within the tumor, though patient (B) showed markedly grater uptake of both agents. The images with ⁶⁴Cu-ATSM had a slightly better target-to background ratio and tumors were delineated more clearly. Reprinted by permission of the Society of Nuclear Medicine from: Lewis JS, Laforest R, Dehdashti F, et al. An Imaging Comparison of 64Cu-ATSM and 60Cu-ATSM in Cancer of the Uterine Cervix. J Nucl Med. 2008; 49(7): 1177–1182. Figure 3 (48)

¹⁸F-misonidazole shows considerable promise in clinical trials, though the rapid decay of F-18 is inconvenient. There have been attempts to correlate the more standard fluorodeoxyglucose (FDG) uptake with hypoxia, but correlations are often poor (50).

Nuclear medicine approaches are costly and less suitable for repeat investigations. Generally, only a single time point is investigated and the measurement is thought to reflect chronic hypoxia, rather than acute hypoxia. For PET, each reporter provides identical signal and thus repeat doses or tandem reporters cannot be readily differentiated except perhaps based on differential decay rates of separate isotopes. Dynamic variations in hypoxia have been assessed, based on biopsy specimens, by applying pairs of hypoxia reporters in a pulse chase fashion with respect to an intervention, as shown by Ljungkvist et al. (33). NMR spectroscopy of ¹⁹F-misonidazoles potentially allows greater versatility, since multiple different reporters could be detected simultaneously based on separate resolved chemical shifts, but it must be noted that NMR requires much higher concentration of reporter molecule (9). An alternative approach is to exploit an MRI reporter, which has physical characteristics influenced by oxygen and detectable by relaxometry as described in the next sections (6).

3.0 Tumor Oximetry: In vivo

While hypoxia may have a somewhat arbitrary definition, oximetry provides quantitative assessment of pO₂. In turn pO₂ can define hypoxic thresholds such as fraction of tumor less than 10 torr (HF₁₀) or HF₅ < 5 torr. It should be noted that various scales are used to report pO₂ [760 torr = 1 atm = 101 kPa], and that pO₂ is related to the concentration of dissolved oxygen [O₂]. It is important to consider the location of the pO₂ measurement, since there are steep gradients from vasculature to interstitium, cytosol and mitochondrion as emphasized by Swartz (51). Ultimately, a parameter must correlate with therapeutic outcome, be reproducible and simple to implement and interpret.

To date, the most extensive evidence for hypoxia in experimental solid tumors is provided by polarographic needle oxygen electrodes. Robust fine needle polarographic electrodes opened the possibility of measuring pO₂ in tumors in situ in vivo to define local pO₂ under baseline conditions or with respect to interventions. Cater and Silver (52) showed the ability to monitor pO₂ at individual locations in patients’ tumors with respect to breathing oxygen. The Eppendorf Histograph has provided the most convincing evidence for hypoxia in human tumors (53–55) and has been considered a “gold standard”, but it is highly invasive. Following extensive studies in animals, the Histograph was used in the clinical setting and has unequivocally revealed hypoxia in many tumor types, e.g., head and neck, cervix, breast, and prostate and even intraoperative deflated lung (2). Moreover, pO₂ distributions have been found to have prognostic value. Disease free survival is significantly worse for patients with hypoxic tumors and applications to cervical cancer have shown correlation between tumor hypoxia and response to irradiation (56). In addition hypoxic tumors have been shown to have poorer response with surgical resection and this is considered to reflect a more aggressive hypoxic phenotype (10, 56, 57). By stepping the electrode through a tumor pO₂ distributions are determined providing a quasi map, though normally presented just in terms of a histogram. Chapman et al. (58) determined that as many as 100 individual locations were required to reproducibly/reliably represent tumor oxygenation. The Eppendorf reveals a pO₂ distribution, which is invariably skewed towards hypoxia. The distribution reported by the histogram reveals heterogeneity, but requires effective data reduction to be useful as a prognostic or predictive biomarker. Variously, mean, median or hypoxic fraction have been reported to correlate with therapeutic response. Hypoxic fraction may be described as HF₁₀, HF₅ or HF_2.5 and may be quoted as a ratio to normal adjoining tissue. The Eppendorf Histograph is not well suited for dynamic measurement of pO₂ with respect to intervention, though repeated measurement in patients with cervical cancer showed that oxygen or carbogen increased tumor oxygenation (59, 60). The Histograph requires tissue access and is impractical in many tumors.

Fiber optic probes can also measure pO₂ directly and are typically finer and do not consume oxygen during measurement. Typically, two or four locations are sampled simultaneously and these optical probes facilitate observation of dynamic changes in pO₂ in response to interventions (61, 62). Fiber optic probes are generally most sensitive to low pO₂ providing little sensitivity above 100 torr.

ESR has been exploited effectively, using both vascular probes and injected implanted particulates (63–65). Most importantly, ESR of implanted carbon chars revealed reoxygenation, in tumors and a correlation with response to irradiation based on measurements at a single location (66). Gallez, et al., investigated many drugs and interventions using ESR and found that several drugs will enhance tumor oxygenation (67).

Materials may be monitored for weeks to months with no apparent deleterious side effects in normal tissue. However, ESR is not readily available for pre-clinical applications and it is essentially impossible in patients due to lack of instrumentation.

3.1 Oximetry Imaging Methods

Given the importance of tumor oxygenation, many diverse methods have been developed and multiple reviews compare and contrast efficiency of imaging approaches. As such, the reader is directed to these articles for historical perspective. In 2004, the NCI sponsored a workshop on hypoxia imaging technologies as summarized in a comprehensive review by Arbeit, et al. (2) featuring contributions from many of the leaders in the field. Imaging approaches, together with needs for the measurements, are extensively described. Other recent reviews have focused on nuclear imaging approaches (8, 17), optical methods (68), ESR (51, 69) and MRI (6, 70). Others have focused on the molecular biology and relevance to therapy and clinical trials (71, 72). In Table 1, we summarize virtues and capabilities of popular methods. In the present review, we will focus on the latest innovations and our own experience with MRI.

In choosing a method, it is crucial to match the capabilities to the problem being addressed. Thus, predicting the efficacy of a hypoxic cell selective cytotoxin may well be ideal using a nitroimidazole reporter, which could exploit optical imaging, MRI or PET (73).

3.2 ¹⁹F MRI

Most MRI oximetry exploits perfluorocarbons (PFCs), where the spin lattice relaxation rate (R₁=1/T₁) is exquisitely sensitive to pO₂ and obeys a linear relationship (R₁= a + b pO₂) at any given magnetic field and temperature. Historically, PFC emulsion blood substitutes were administered systemically (74). Following iv administration, they exhibit prolonged vascular circulation and can provide measurements of vascular pO₂ for several hours, though measurements are potentially susceptible to flow artifacts. Ultimately, PFCs become sequestered in tissue, primarily in the reticuloendothelial system (RES, liver, spleen and bone marrow) allowing pO₂ measurements (74). Small amounts are found in myocardium and tumor and have been exploited for oximetry. The early PFC emulsions (Fluosol^TM; Oxypherol^TM (perfluorotributylamine); Oxygent^TM (perfluorooctylbromide); Therox^TM (F44E)) had multiple resonances and the requirement for chemical shift selective imaging often caused signal loss. However, each signal showed unique sensitivity to pO₂ and temperature and this phenomenon could be exploited to determine both parameters simultaneously (75). Systemic administration had the great advantage of being non-invasive, but most of the small fraction of PFC, which became sequestered in the tumor was found in well perfused tumor regions biasing measurements (76).

Symmetrical PFCs may have a single ¹⁹F resonance offering better signal to noise ratio. Fifteen-crown-five-ether has been used as an emulsion to probe liver and tumor pO₂ (77). Hexafluorobenzene (HFB) provides an alternative, which is more readily commercially available, more sensitive to changes in pO₂ and less sensitive to changes in temperature (6). Hexafluorobenzene has an extremely high vapor pressure bringing potential benefits or problems. It does not form stable biocompatible emulsions and requires direct injection into tissue. It also clears rapidly so that pO₂ measurements are generally only possible on the day of administration. Direct injection into a tissue has the distinct advantage that any region of interest may be interrogated immediately without needing to wait for vascular clearance. Moreover, there are no concerns about potential toxicity related to long-term retention. Hexafluorobenzene exhibits an extremely broad range of T₁ from 0.7 s to 13 s at 4.7 Tesla, so that imaging may be slow. Since signal to noise ratio (SNR) influences the precision of pO₂ measurements, the benefits of HFB may be less than anticipated (78). The potential long acquisition times required to assess long T₁s were overcome by applying echo planar imaging (EPI). We routinely achieve pO₂ maps with 50 to 150 individual locations simultaneously in 6½ minutes in rat tumors with a precision of 1 to 3 torr in hypoxic regions following administration of 50 μl HFB. We have used the so-called FREDOM approach (Fluorocarbon Relaxometry using Echo planar imaging for Dynamic Oxygen Mapping) to examine multiple tumor types implanted in rats and mice including breast, lung and prostate. Representative images and data are shown in Figures 4–7 and Table 3. As expected, median pO₂ and hypoxic faction observed in rat tumors were inversely related (Figure 5).

Figure 4. Heterogeneity of tumor oxygenation.

Maps of pO₂ were generated using FREDOM following direct injection of 100 μl hexafluorobenzene into the respective Dunning R3327 prostate tumors growing syngeneically on thigh of Copenhagen rats. Each tumor shows a range of pO₂s, which are presented as histograms below. Sequential pO₂ maps were acquired over 20 mins, while anesthetized rats breathed air +1% isoflurane and the baseline stability is revealed by the consistent histograms. For the fast growing small MAT-Lu tumor (A, D: volume 1.2 cm³; volume doubling time ~2 days) mean pO₂=22.1 torr, median pO₂= 19.8 torr and HF₁₀= 40%. For the moderately well differentiated large HI tumor (B, E: volume 7.6 cm³; volume doubling time ~10 days) mean pO₂= 13.4 torr, median pO₂= 13.4 torr and HF₁₀= 76%. For the slow growing small H tumor (C, F: volume 0.6; volume doubling time ~20 days) mean pO₂= 36.9 torr, median pO₂= 37.5 torr and HF₁₀= 8%. G shows the cumulative hypoxic fraction of each of the tumors MAT-Lu (green); HI (blue) and H (red). These data are quite consistent with the population mean values reported in Table 3. Relaxation curves for representative voxels H T₁=11.61 s (pO₂=0.78 torr) and I T₁=1.47 s (pO₂=46.5 torr).

Figure 7. Tumor oxygenation with respect to vascular targeting agent bavituximab.

Hexafluorobenzene (50 μl) was injected directly into a small Dunning prostate R3327-AT1 tumor (0.94 cm³) and pO₂ maps were obtained using FREDOM in 6.5 mins each. Following a) baseline measurement, bavituximab was injected (0.35 ml intravenously) and further pO₂ maps were acquired over the following 2 hrs (b after 30 mins and c after 120 min). d) Mean pO₂ values + standard deviation are plotted (d) showing little acute response.

Table 3.

CHARACTERISTICS OF TUMOR SUBLINES

Tumor Dunning prostate R3327 lines	VDT (volume doubling time; days)	Hypoxic fraction HF10 (%)		Mean pO2 (torr)		Response to oxygen breathing
		Small	Large	Small	Large
MAT-Lu* (84)	2.7	23 14	58 40	24 60	8 27	Slow
AT1 air O2 (81)	5	61 24	83 59	10 62	4 40	Slow
HI (84, 85)	9	20 15*	80 20	29 75*	2 102	Rapid
H air O2 (81)	16	16 3	46 10	34 137	13 83	Moderate

Small tumors <2 cm³; large >3.5 cm³. AT1, HI, and H tumors on rats breathing isoflurane/air;

^*older anesthesia protocol using methoxyflurane with 33% O₂/66%N₂O for baseline.

Figure 5. Tumor oxygenation assessed by ¹⁹F MRI.

A) Relationships between median pO₂ in Dunning prostate R3327 pedicle tumors and hypoxia faction. MAT-Lu (pink); AT1 (blue); HI (green); H (black). As expected a generally inverse relationship was observed based on FREDOM.

B) Baseline pO₂ was measured during air breathing and is related to maximum median pO₂ observed during a hyperoxic gas breathing challenge with oxygen. For any given tumor type a reasonable correlation was found, e.g., pink and blue lines, but each tumor type behaved very differently.

We have applied this approach to multiple studies and have demonstrated measurements commensurate with more traditional techniques such as polarographic electrodes (including the Eppendorf Histograph) (79, 80), optical fiber probes (62), immunohistochemistry (81), and near-infrared spectroscopy (82), in terms of baseline pO₂ and response to interventions.

The greatest strength of ¹⁹F MRI is the ability to make sequential measurements over a period of hours with respect to interventions. Maps of pO₂ allow individual tumor regions to be monitored or cluster analysis reveals differential behavior for regions showing shared baseline characteristics. In some cases, the baseline measurement is indicative of response to intervention such as breathing hyperoxic gas (Figure 5b). For most tumors, we find that initially well oxygenated tumor regions show a particularly large response to hyperoxic gas breathing (83). In virtually every mouse and rat tumor system examined to date, we found progressive hypoxiation with increased size (Table 3 + Figure 6) (62, 81, 84). Small tumors are generally well oxygenated with small hypoxic fractions, but somewhere between 1 and 3 cm³ a catastrophic decline occurs in oxygen tension (62, 81, 84).

Figure 6. Modulating tumor response to irradiation in Dunning prostate R3327-HI tumors.

The Kaplan Meier hazard plots indicate time to reach 2 x initial size (T₂).

Bottom: Large HI tumors exhibit extensive hypoxia (histogram, bottom left), however it is essentially eliminated by breathing oxygen (histogram, top left) resulting in excellent response to irradiation. Here a single dose of 30 Gy (half-the TCD₅₀) was applied and the time to double in size was observed. Tumors on rats breathing air during irradiation showed little benefit over controls. By contrast, a significant growth delay (51 days; p < 0.01) was observed in large HI tumors, when rats breathed oxygen 30 min prior to and during irradiation. Baseline hypoxic fraction was high HF₁₀>65%. However, breathing oxygen essentiality eliminated the hypoxic fraction (HF₁₀<20%) and these tumors showed a significant growth delay compared with controls or irradiated air breathing animals.

Top: Small HI tumors, which are well oxygenated, respond to irradiation. Here, a single dose of 30 Gy (half-the TCD₅₀) was applied and the time to double in size was observed. Tumors on rats breathing air (black line) or oxygen (red line) showed a significant growth delay compared with controls (green line). Baseline hypoxic fraction was small HF₁₀<25%. Modified from (85).

It is well documented that hypoxia leads to radiation resistance. During a course of conventional fractionated radiotherapy, progressive re-oxygenation is reported to occur. However, for a single dose or limited sequential doses, hypoxic fraction may be far more important. We have shown that irradiation (single dose 30 Gy) of large hypoxic Dunning prostate R3327-HI tumors caused limited tumor growth delay (Figure 6). However, this tumor type shows a pronounced oxygenation accompanying hyperoxic gas breathing, essentially eliminating the hypoxic fraction (HF₁₀ < 20%; Table 3). With oxygen breathing there was a significant tumor growth delay accompanying the same single dose of irradiation (85). By contrast, in small HI tumors, where the HF₁₀ <20 torr for air breathing, there was a significant tumor growth delay accompanying irradiation and no additional benefit was seen for breathing oxygen (Figure 6). We note that large AT1 tumors, which exhibit a high hypoxic fraction that resists modulation also show minimal benefit from breathing oxygen (86). Thus, measurements of baseline pO₂ may be indicative of radiation response and assessment of the ability to manipulate tumor oxygenation using an intervention may be relevant to predicting such adjuvant interventions for enhancing radiation. Noting that some tumors resist hyperoxic gas breathing, it is tempting to apply hyperbaric oxygen and indeed using fiber optic probes, we showed that hypoxic regions of tumors, which resisted modulation by 100% normobaric oxygen, did respond to hyperbaric oxygen (87). We have previously shown that vasoactive drugs, such as hydralazine caused reduced tumor oxygenation (88). As expected, the vascular disrupting agent Combretastatin A4P caused rapid hypoxiation (89) and preliminary data show that this strongly influences the efficacy of combined radiation therapy. Meanwhile, the vascular targeting agent, bavituximab, has a minimal acute effect on pO₂ over a period of two hours (Figure 7).

3.3 ¹H MRI

For small animal studies, quantitative ¹⁹F MRI oximetry of hexafluorobenzene (HFB) is very effective. There is no background signal and single resonance provides very high sensitivity to pO₂. However, we have been exploring proton MRI alternatives with view to greater translatability to the clinical setting. Noting the requisite properties of the ¹⁹F perfluorocarbon reporters (hydrophobic, liquid, single resonance, unreactive, high gas solubility), we sought analogous proton agents and identified hexamethyldisiloxane (HMDSO) (90). Detection does require fat and water suppression, but this is routinely achieved for imaging metabolites such as N-acetyl aspartate, lactate, choline and citrate in brain or prostate of patients. Thus, proton magnetic resonance spectroscopy (90) or magnetic resonance imaging (91) may be used analogously to FREDOM (6) and we have initiated investigations using HMDSO (e.g., PISTOL: Proton Imaging of Siloxanes to map Tissue Oxygenation Levels). We show an example of the detection of hypoxiation following administration of the vascular disrupting agent Combretastatin (CA4P ) to a prostate tumor bearing rat in Figure 8 and this closely matches previous ¹⁹F MRI results (89).

Figure 8. Monitoring oxygenation changes with respect to vascular targeting agent Combretastatin A4-phosphate using PISTOL.

HMDSO (100 μl) was injected directly into Dunning R3327-H prostate tumor implanted in a Copenhagen rat. Sequential pO₂ maps were measured before and for 24 hrs following the administration of the vascular disrupting agent Combretastatin A4-phosphate (CA4P; 30 mg/kg). Upper panel shows images on day 1, while the lower panel shows pO₂ dynamics and response to oxygen breathing at 2 and 24 hrs post administration of CA4P compared to baseline (CA4P kindly provided by OxiGene).

4.0 Endogenous reporters

Many reporter molecules are routinely exploited with diverse imaging modalities to measure tumor hypoxia or pO₂. Ideally, oxygenation could be related to endogenous characteristics. Since many biochemical pathways are under oxygen regulation, they can provide an elegant window on hypoxia, e.g., induction of HIF-1 and Glut-1 together with secondary responses, such as increased production of VEGF, NIP3 and tumor associated macrophage activity. However, assessment of expression requires biopsy. Additionally, the optical absorption spectra of oxy- and deoxyhemoglobin are quite different allowing interrogation of vascular oxygenation by spectroscopy and imaging though spatial resolution is generally quite poor (68, 82). Deoxyhemoglobin is also paramagnetic and forms the basis of BOLD MRI.

4.1 BOLD (blood oxygen level dependent) contrast ¹H MRI

BOLD contrast depends on deoxyhemoglobin, which is paramagnetic. Deoxygenated red blood cells generate local magnetic field gradients causing signal dephasing, which appears as T₂* relaxation and can be detected as signal loss in T₂*-weighted images. The magnitude of the effect depends on hematocrit, vascular volume, and concentration of deoxyhemoglobin. Images may further be influenced by flow effects leading to the concept of FLOOD (FLow and Oxygen Level Dependant) (92). The BOLD effect is widely exploited in studies of brain activation, where stimuli generate regional changes in blood flow, altering deoxyhemoglobin concentration and generating contrast, which is the foundation of fMRI (93). In blood, a direct relationship is found between the T₂* and pO₂ based on the sigmoidal O₂ binding curve of hemoglobin. For large blood vessels T₂* may be used to measure vascular oxygenation, and hence pO₂, directly (94) and a recent development in the brain is sometimes referred to as qBOLD (95).

Interpretation of BOLD in tumors is more complex due to the bed of tortuous small capillaries, such that voxels include both blood vessels and surrounding tissue. Several groups have examined the effect and its relationship to tumor oxygenation, notably Howe, Robinson and Griffiths at St George’s, London (92, 96, 97). A direct correlation between pO₂ and the BOLD signal changes has been found in several studies. Al Hallaq et al. (98) reported a strong linear correlation between change in pO₂ measured by electrode and change in the signal line widths (essentially T₂*). Elas et al. and Dunn et al. compared EPR (electron paramagnetic resonance) estimates of oxygenation and BOLD response and each found consistent data (65, 99). We recently showed a strong correlation between changes in T₂*-weighted signal and pO₂ based on ¹⁹F MR oximetry (100). Perhaps most significantly, we found that a large BOLD response (>2% ΔSI) to hyperoxic gas challenge (carbogen) corresponded with essential elimination of hypoxic fraction in rat breast tumors. Meanwhile, a small BOLD response generally indicated larger residual hypoxic fraction resistant to manipulation. Baudelet and Gallez reviewed BOLD investigations of tumors thoroughly (101) and also reported correlation of BOLD signal changes versus fiber optic oxygen tension measurements and oxygen challenge (102). The results indicated that there was always a positive correlation, but that a 10% change in relative signal intensity in the BOLD experiment could correspond to an increase in pO₂ < 25 torr or > 100 torr. We contend that either change would be radiobiologically pertinent, because once pO₂ exceeds 10 torr there is relatively little further oxygen enhancement achieved by increased levels of oxygen.

Currently, Padhani et al. are pursuing the use of BOLD to investigate tumors at various sites in patients with an emphasis on prostate cancer (103–105). Their primary method differs somewhat from our approach in that they assess baseline R₂* and relate it to inherent hypoxia rather than evaluating hypoxia based on the response to an oxygen breathing challenge. Rodrigues et al., showed that R₂* itself was indicative of hypoxia and correlated with response to radiation in tumors (106).

We note the BOLD response reflects vascular oxygenation, which may be disconnected from tissue pO₂. While increased oxygen delivery is expected to elevate local pO₂, the additional oxygen could be consumed, as noted by Gullino and Vaupel in studies of perfused tumors (107, 108). Thus, we believe it is imperative to also include an assessment of tissue oxygenation (pO₂).

4.2 TOLD (Tissue oxygen level dependant) contrast ¹H MRI

Molecular oxygen (O₂) is paramagnetic and hence [O₂] influences tissue water spin-lattice relaxation (R₁). Recently, Matsumoto et aI. (109) demonstrated T₁ sensitive signal response in tumors to breathing hyperbaric oxygen, which reflects changes in tissue pO₂. Others have explored normal tissue response to hyperoxic gas breathing, notably Edelman et al. (110) and recently O’Connor et al. (111, 112), Jones et al. (113) and Tademura et al. (114). However, except in rare circumstances, such as vitreous humor or CSF, it is probably not a suitable measure of absolute pO₂, since many factors can alter R₁ (115, 116). We believe it can provide suitable evaluation of acute changes in pO₂.

We believe that combining BOLD and TOLD response to hyperoxic gas intervention provides particularly robust insight into tumor hypoxia and potential modulation. This is the basis of the simple test DOCENT (Dynamic Oxygen Challenge Evaluated by NMR T₁ and T₂*), as shown in Figure 9. To demonstrate the method, we chose a small HI tumor, which is known to be particularly well oxygenated and exhibit a large response to breathing hyperoxic gas (Table 3). We note a stable baseline for BOLD and a rapid response in signal intensity approaching 40% ΔSI within one minute of breathing carbogen (95% O₂, 5% CO₂) (Figure 9). Distinct heterogeneity was observed in any given slice, but quite similar data were seen by MRI in three adjacent tissue slices. The TOLD response was smaller and more sluggish, as expected, since O₂ must diffuse from the vasculature into the tumor tissue. Importantly, the combined methods robustly demonstrate delivery and accumulation of oxygen. It remains to be seen whether quantitative R₁ and R₂* measurements are required or simple R₁- and R₂*-weighted signal intensity changes will be useful. We are currently evaluating whether such measurements are predictive for radiation response in rat tumors. We are also implementing these methods in patients.

Figure 9. DOCENT.

Sequential interleaved T₂*-weighted (BOLD) and T₁-weighted (TOLD) 1H MR images were obtained from HI tumor (0.45 cm³) during baseline air breathing (a BOLD and d TOLD, respectively) and with respect to carbogen challenge (b and e for BOLD and TOLD respectively). c) within 2 minutes there was a large significant BOLD response reaching a plateau of about 40% signal enhancement indicating reduced levels of vascular deoxyhemoglobin. f) a more sluggish TOLD response was seen over several minutes consistent with delivery of oxygen to the tissues and elevated pO₂. Heterogeneity of signal response was seen within the tumor slice, but three adjacent slices showed remarkably consistent response (green, red and blue lines show mean response for each slice).

5 Innovations and latest applications

Within the past year, several innovations have been presented promising new insight into tumor hypoxia. There has been notable progress in quantitative oximetry based on ¹⁹F MRI with new reports of applications. Gallez et al. implemented a faster imaging approach for ¹⁹F MRI of hexafluorobenzene, Look-Locker type SNAP-IR measurements giving 90 s time resolution (117, 118). Spatial resolution was similar to that previously reported for FREDOM (requiring 6.5 mins), though a larger volume of hexafluorobenzene reporter molecule was used (90 vs. 50 μl) and it remains to be seen whether the precision of measurements will be equivalent, since SNR is known to strongly influence the quality of relaxation rate measurements (78). We do ourselves sometimes use larger volumes of HFB in tumors, e.g. Figure 4. In any case, the faster temporal resolution opens new opportunities to explore rapid dynamic pO₂ fluctuations and transients: a new window on acute hypoxic episodes. Notably, both the groups in Louvain and UCSF are using ¹⁹F MR oximetry of hexafluorobenzene to explore oxygen dynamics in response to pharmaceutical interventions (119–121). The use of HFB as a ¹⁹F pO₂ reporter molecule is gaining popularity with new reports of applications. Diepart et al. have further used this technique to measure oxygen consumption in tumors (120). Using the FREDOM technique, Liu et al. (122) compared pO₂ changes in multiple organs during isovolemic anemic hemodilution using hemoglobin based oxygen carriers under normoxic and hyperoxic conditions in a rat model.

While we favor direct intra tumoral injection of HFB for immediate interrogation of regions of interest, we note a novel approach presented by Ahrens et al. (123). Several groups have pre-labeled cells in culture with PFC to allow cell tracking after implantation in vivo (124) and this has now been used to measure changes in intracellular pO₂ following implantation of cells.

Interrogation of hypoxia by NMR has received new impetus with the presentation of a trifluoronitroimidazole, which provided sufficient signal to noise to allow chemical shift imaging, as opposed to the more traditional signal limited spectroscopy (125). A novel approach uses a gadolinium liganded nitroimidazole (GdDO3NI), which showed increased MRI contrast in hypoxic central regions of rat prostate AT1 tumors compared to peripheral, well-perfused regions. Increased uptake of GdDO3NI in the tumor center was further confirmed by flame ionization spectroscopy post mortem (126).

Well characterized animal models exhibiting differential hypoxia are important for evaluating new methods of detecting hypoxia and evaluating novel therapies. Based on ¹⁹F MRI, we have extensively characterized a series of Dunning prostate R3327 rat tumors, which exhibit a wide range of oxygen characteristics, as shown in Table 3. The Dunning prostate tumors have the great advantage of being syngeneic in rats allowing investigations in immunocompetent animals. Moreover, rats show much more stable physiology than mice under anesthesia and greater tumor burden is feasible. Indeed, knowledge of the hypoxia status was crucial in choosing a model to evaluate GdDO3NI

We find three distinct characteristics for tumor oxygenation and dynamics:

1. hypoxic tumors, which do not respond to hyperoxic gas intervention;

2. hypoxic tumors, which do respond to hyperoxic gas intervention (e.g., large HI, Figure 6);

3. well oxygenated tumors (e.g., small HI, Figure 6). In every case examined to date, well oxygenated tumors became even better oxygenated with hyperoxic gas breathing.

6. Conclusions

Increasingly, there is evidence that hypoxia influences angiogenesis, tumor invasion and metastasis. Moreover, repeated bouts of intermittent hypoxic stress may be important in stimulating tumor progression. Thus, the ability to assess tumor oxygenation non-invasively, and repeatedly, with respect to acute or chronic interventions becomes increasingly important.

PET approaches are costly for small animal research, but they have already made the transition to clinical trials. They do require reporter molecules and suffer from the complications of radioactivity, notably substrate decay and waste disposal.

MRI approaches are very attractive for pre-clinical investigations of hypoxia and oxygen dynamics. They offer high spatial resolution, arbitrary tissue depth penetration and avoids complications of radioactivity. However, data acquisition is not trivial, throughput is quite modest and instrumentation is expensive. Uniquely, MRI can provide quantitative maps of pO₂ and reveal dynamic changes in response to interventions based on inert reporter molecules. ¹⁹F MR oximetry, e.g., FREDOM, based on hexafluorobenzene is perfectly suited to measurements in pre-clinical animal studies providing quantitative pO₂ measurements with useful spatial and temporal resolution and precision relevant to radiotherapy (6). Given the well documented lack of toxicity clinical application ultimately be feasible, but currently ¹⁹F MRI remains esoteric on clinical MR scanners. PISTOL offers an analogous proton MRI approach (91). Repeat measurements of pO₂ are non-invasive, but both PISTOL and FREDOM approaches do require a reporter molecule.

Meanwhile, BOLD and TOLD MRI approaches offer entirely non-invasive assessments of tissue oxygenation dynamics and are clearly preferable for clinical applications. While they do not provide quantitative measurements of pO₂ there is increasing evidence that changes in R₁ and R₂* relate to changes in tissue and vascular oxygenation. Together the offer inter-dependant insight into tumor oxygenation and DOCENT (Dynamic Oxygen Challenge Evaluated by NMR T₁ and T₂*) promises to become a test for human tumor hypoxia and response to intervention based on the simple act of breathing oxygen during MRI.