Journal of Radiology and Radiation Therapy

Bench-To-Bedside Oximetry for Real-Time Monitoring of Tumor Po2 : A Critical Parameter Which Influences Radiotherapeutic Outcome

Mini Review | Open Access

  • 1. EPR Center for the Study of Viable Systems, Department of Radiology, USA
  • 2. Department of Surgery Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA
  • 3. Department of Medicine, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA
  • 4. Norris Cotton Cancer Center, One Medical Center Drive, Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756, USA
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Corresponding Authors
Periannan Kuppusamy, Geisel School of Medicine at Dartmouth, 48 Lafayette Street, Lebanon, NH 03766, Tel: 603-650-1034

1. Moeller BJ, Richardson RA, Dewhirst MW. Hypoxia and radiotherapy: opportunities for improved outcomes in cancer treatment. Cancer Metastasis Rev. 2007; 26: 241-248.

2. Hall E. Radiobiology for the radiologist. 6th ed. Philadelphia: Lippincott Williams & Wilkins; 2006.

3. Vaupel P, Mayer A. Hypoxia in cancer: significance and impact on clinical outcome. Cancer Metastasis Rev. 2007; 26: 225-239.

4. Overgaard J. Hypoxic radiosensitization: adored and ignored. J Clin Oncol. 2007; 25: 4066-4074. 5. Vaupel P, Mayer A, Höckel M. Tumor hypoxia and malignant progression. Methods Enzymol. 2004; 381: 335-354.

6. Weinmann M, Belka C, Plasswilm L. Tumour hypoxia: impact on biology, prognosis and treatment of solid malignant tumours. Onkologie. 2004; 27: 83-90.

7. Vaupel P. Hypoxia and aggressive tumor phenotype: implications for therapy and prognosis. Oncologist. 2008; 13 Suppl 3: 21-26.

8. Brizel DM, Dodge RK, Clough RW, Dewhirst MW. Oxygenation of head and neck cancer: changes during radiotherapy and impact on treatment outcome. Radiother Oncol. 1999; 53: 113-117.

9. Nordsmark M, Bentzen SM, Rudat V, Brizel D, Lartigau E, Stadler P, et al. Prognostic value of tumor oxygenation in 397 head and neck tumors after primary radiation therapy. An international multi-center study. Radiother Oncol. 2005; 77: 18-24.

10. Brizel DM, Sibley GS, Prosnitz LR, Scher RL, Dewhirst MW. Tumor hypoxia adversely affects the prognosis of carcinoma of the head and neck. Int J Radiat Oncol Biol Phys. 1997; 38: 285-289.

11. Brizel DM, Rosner GL, Prosnitz LR, Dewhirst MW. Patterns and variability of tumor oxygenation in human soft tissue sarcomas, cervical carcinomas, and lymph node metastases. Int J Radiat Oncol Biol Phys. 1995; 32: 1121-1125.

12. Begg AC, Janssen H, Sprong D, Hofland I, Blommestijn G, Raleigh JA, et al. Hypoxia and perfusion measurements in human tumors--initial experience with pimonidazole and IUdR. Acta Oncol. 2001; 40: 924- 928.

13. Lyng H, Sundfør K, Rofstad EK. Changes in tumor oxygen tension during radiotherapy of uterine cervical cancer: relationships to changes in vascular density, cell density, and frequency of mitosis and apoptosis. Int J Radiat Oncol Biol Phys. 2000; 46: 935-946.

14. Cooper RA, West CM, Logue JP, Davidson SE, Miller A, Roberts S, et al. Changes in oxygenation during radiotherapy in carcinoma of the cervix. Int J Radiat Oncol Biol Phys. 1999; 45: 119-126.

15. Khan N, Li H, Hou H, Lariviere JP, Gladstone DJ, Demidenko E, et al. Tissue pO2 of orthotopic 9L and C6 gliomas and tumor-specific response to radiotherapy and hyperoxygenation. Int J Radiat Oncol Biol Phys. 2009; 73: 878-885.

16. Olive PL. Radiation-induced reoxygenation in the SCCVII murine tumour: evidence for a decrease in oxygen consumption and an increase in tumour perfusion. Radiother Oncol. 1994; 32: 37-46.

17. Fujii H, Sakata K, Katsumata Y, Sato R, Kinouchi M, Someya M, et al. Tissue oxygenation in a murine SCC VII tumor after X-ray irradiation as determined by EPR spectroscopy. Radiother Oncol. 2008; 86: 354- 360.

18. Khan N, Williams BB, Hou H, Li H, Swartz HM. Repetitive tissue pO2 measurements by electron paramagnetic resonance oximetry: current status and future potential for experimental and clinical studies. Antioxid Redox Signal. 2007; 9: 1169-1182.

19. Ahmad R, Kuppusamy P. Theory, instrumentation, and applications of electron paramagnetic resonance oximetry. Chem Rev. 2010; 110: 3212-3236.

20. Gallez B, Baudelet C, Jordan BF. Assessment of tumor oxygenation by electron paramagnetic resonance: principles and applications. NMR Biomed. 2004; 17: 240-262.

21. Khan N, Mupparaju S, Hekmatyar SK, Hou H, Lariviere JP, Demidenko E, et al. Effect of hyperoxygenation on tissue pO2 and its effect on radiotherapeutic efficacy of orthotopic F98 gliomas. Int J Radiat Oncol Biol Phys. 2010; 78: 1193-1200.

22. Hou H, Li H, Dong R, Mupparaju S, Khan N, Swartz H. Cerebral oxygenation of the cortex and striatum following normobaric hyperoxia and mild hypoxia in rats by EPR oximetry using multiprobe implantable resonators. Adv Exp Med Biol. 2011; 701: 61-67.

23. Hou H, Lariviere JP, Demidenko E, Gladstone D, Swartz H, Khan N. Repeated tumor pO(2) measurements by multi-site EPR oximetry as a prognostic marker for enhanced therapeutic efficacy of fractionated radiotherapy. Radiother Oncol. 2009; 91: 126-131.

24. Hou H, Dong R, Li H, Williams B, Lariviere JP, Hekmatyar SK, et al. Dynamic changes in oxygenation of intracranial tumor and contralateral brain during tumor growth and carbogen breathing: a multisite EPR oximetry with implantable resonators. J Magn Reson. 2012; 214: 22-28.

25. Hou H, Dong R, Lariviere JP, Mupparaju SP, Swartz HM, Khan N. Synergistic combination of hyperoxygenation and radiotherapy by repeated assessments of tumor pO2 with EPR oximetry. J Radiat Res. 2011; 52: 568-574.

26. Khan N, Mupparaju S, Hou H, Williams BB, Swartz H. Repeated assessment of orthotopic glioma pO(2) by multi-site EPR oximetry: a technique with the potential to guide therapeutic optimization by repeated measurements of oxygen. J Neurosci Methods. 2012; 204: 111-117.

27. Ilangovan G, Zweier JL, Kuppusamy P. Mechanism of oxygen-induced EPR line broadening in lithium phthalocyanine microcrystals. J Magn Reson. 2004; 170: 42-48.

28. Liu KJ, Gast P, Moussavi M, Norby SW, Vahidi N, Walczak T, et al. Lithium phthalocyanine: a probe for electron paramagnetic resonance oximetry in viable biological systems. Proc Natl Acad Sci U S A. 1993; 90: 5438-5442.

29. Pandian RP, Parinandi NL, Ilangovan G, Zweier JL, Kuppusamy P. Novel particulate spin probe for targeted determination of oxygen in cells and tissues. Free Radic Biol Med. 2003; 35: 1138-1148.

30. Ilangovan G, Manivannan A, Li H, Yanagi H, Zweier JL, Kuppusamy P. A naphthalocyanine-based EPR probe for localized measurements of tissue oxygenation. Free Radic Biol Med. 2002; 32: 139-147.

31. Meenakshisundaram G, Eteshola E, Pandian RP, Bratasz A, Selvendiran K, Lee SC, et al. Oxygen sensitivity and biocompatibility of an implantable paramagnetic probe for repeated measurements of tissue oxygenation. Biomed Microdevices. 2009; 11: 817-826.

32. Pandian RP, Meenakshisundaram G, Bratasz A, Eteshola E, Lee SC, Kuppusamy P. An implantable Teflon chip holding lithium naphthalocyanine microcrystals for secure, safe, and repeated measurements of pO2 in tissues. Biomed Microdevices. 2010; 12: 381-387.

33. Meenakshisundaram G, Pandian RP, Eteshola E, Lee SC, Kuppusamy P. A paramagnetic implant containing lithium naphthalocyanine microcrystals for high-resolution biological oximetry. J Magn Reson. 2010; 203: 185-189.

34. Meenakshisundaram G, Eteshola E, Pandian RP, Bratasz A, Lee SC, Kuppusamy P. Fabrication and physical evaluation of a polymer-encapsulated paramagnetic probe for biomedical oximetry. Biomed Microdevices. 2009; 11: 773-782.

35. Dinguizli M, Jeumont S, Beghein N, He J, Walczak T, Lesniewski PN, et al. Development and evaluation of biocompatible films of polytetrafluoroethylene polymers holding lithium phthalocyanine crystals for their use in EPR oximetry. Biosens Bioelectron. 2006; 21: 1015-1022.

36. Salikhov I, Walczak T, Lesniewski P, Khan N, Iwasaki A, Comi R, et al. EPR spectrometer for clinical applications. Magn Reson Med. 2005; 54: 1317-1320.

37. Williams BB, Hou H, Grinberg OY, Demidenko E, Swartz HM. High spatial resolution multisite EPR oximetry of transient focal cerebral ischemia in the rat. Antioxid Redox Signal. 2007; 9: 1691-1698.

38. Grinberg VO, Smirnov AI, Grinberg OY, Grinberg SA, O’Hara JA, Swartz HM. Practical Experimental Conditions and Limitations for HighSpatial-Resolution Multisite EPR Oximetry. Appl Magn Reson. 2005; 28: 69-78.

39. Ahmad R, Caia G, Potter LC, Petryakov S, Kuppusamy P, Zweier JL. In vivo multisite oximetry using EPR-NMR coimaging. J Magn Reson. 2010; 207: 69-77.

40. Khan N, Mupparaju SP, Mintzopoulos D, Kesarwani M, Righi V, Rahme LG, et al. Burn trauma in skeletal muscle results in oxidative stress as assessed by in vivo electron paramagnetic resonance. Mol Med Rep. 2008; 1: 813-819.

41. Khan N, Mupparaju SP, Hou H, Lariviere JP, Demidenko E, Swartz HM, et al. Radiotherapy in conjunction with 7-hydroxystaurosporine: a multimodal approach with tumor pO2 as a potential marker of therapeutic response. Radiat Res. 2009; 172: 592-597.

42. Khan N, Khramtsov V, Swartz H, editors. Methods for Tissue pO2, Redox, pH, and Glutathione Measurements by EPR Spectroscopy Mary Ann Liebert, Inc.; 2010.

43. Hou H, Khan N, Grinberg OY, Yu H, Grinberg SA, Lu S, et al. The effects of Efaproxyn (efaproxiral) on subcutaneous RIF-1 tumor oxygenation and enhancement of radiotherapy-mediated inhibition of tumor growth in mice. Radiat Res. 2007; 168: 218-225.

44. Hou H, Abramovic Z, Lariviere JP, Sentjurc M, Swartz H, Khan N. Effect of a topical vasodilator on tumor hypoxia and tumor oxygen guided radiotherapy using EPR oximetry. Radiat Res. 2010; 173: 651-658.

45. Helisch A, Wagner S, Khan N, Drinane M, Wolfram S, Heil M, et al. Impact of mouse strain differences in innate hindlimb collateral vasculature. Arterioscler Thromb Vasc Biol. 2006; 26: 520-526.

46. Doloff JC, Khan N, Ma J, Demidenko E, Swartz HM, Jounaidi Y. Increased tumor oxygenation and drug uptake during anti-angiogenic weekly low dose cyclophosphamide enhances the anti-tumor effect of weekly tirapazamine. Curr Cancer Drug Targets. 2009; 9: 777-788.

47. Friedman BJ, Grinberg OY, Isaacs KA, Ruuge EK, Swartz HM. Effect of repetitive ischemia on myocardial oxygen tension in isolated perfused and hypoperfused rat hearts. Magn Reson Med. 1996; 35: 214-220.

48. Grinberg OY, Friedman BJ, Swartz HM. Intramyocardial pO2 measured by EPR. Adv Exp Med Biol. 1997; 428: 261-268.

49. Grinberg OY, Grinberg SA, Friedman BJ, Swartz HM. Myocardial oxygen tension and capillary density in the isolated perfused rat heart during pharmacological intervention. Adv Exp Med Biol. 1997; 411: 171-181.

50. Khan M, Kwiatkowski P, Rivera BK, Kuppusamy P. Oxygen and oxygenation in stem-cell therapy for myocardial infarction. Life Sci. 2010; 87: 269-274.

51. Mupparaju S, Hou H, Lariviere JP, Swartz H, Jounaidi Y, Khan N. Repeated tumor oximetry to identify therapeutic window during metronomic cyclophosphamide treatment of 9L gliomas. Oncology reports. 2011; 26: 281-286.

52. Romiti A, Cox MC, Sarcina I, Di Rocco R, D’Antonio C, Barucca V, et al. Metronomic chemotherapy for cancer treatment: a decade of clinical studies. Cancer Chemother Pharmacol. 2013; 72: 13-33.

53. Pasquier E, Kavallaris M, André N. Metronomic chemotherapy: new rationale for new directions. Nat Rev Clin Oncol. 2010; 7: 455-465.

54. McGee MC, Hamner JB, Williams RF, Rosati SF, Sims TL, Ng CY, et al. Improved intratumoral oxygenation through vascular normalization increases glioma sensitivity to ionizing radiation. Int J Radiat Oncol Biol Phys. 2010; 76: 1537-1545.

55. Mupparaju S, Hou H, Lariviere JP, Swartz HM, Khan N. Tumor pOâ‚‚ as a surrogate marker to identify therapeutic window during metronomic chemotherapy of 9L gliomas. Adv Exp Med Biol. 2011; 701: 107-113.

56. Li H, Hou H, Sucheta A, Williams BB, Lariviere JP, Khan MN, et al. Implantable resonators--a technique for repeated measurement of oxygen at multiple deep sites with in vivo EPR. Adv Exp Med Biol. 2010; 662: 265-272.

57. O’Hara JA, Hou H, Demidenko E, Springett RJ, Khan N, Swartz HM. Simultaneous measurement of rat brain cortex PtO2 using EPR oximetry and a fluorescence fiber-optic sensor during normoxia and hyperoxia. Physiol Meas. 2005; 26: 203-213.

58. Dietrich J, Norden AD, Wen PY. Emerging antiangiogenic treatments for gliomas - efficacy and safety issues. Curr Opin Neurol. 2008; 21: 736-744.

59. Tran Cao HS, Bouvet M, Kaushal S, Keleman A, Romney E, Kim G, et al. Metronomic gemcitabine in combination with sunitinib inhibits multisite metastasis and increases survival in an orthotopic model of pancreatic cancer. Mol Cancer Ther. 2010; 9: 2068-2078.

60. Laquente B, Lacasa C, Ginestà MM, Casanovas O, Figueras A, Galán M, et al. Antiangiogenic effect of gemcitabine following metronomic administration in a pancreas cancer model. Mol Cancer Ther. 2008; 7: 638-647.

61. Cham KK, Baker JH, Takhar KS, Flexman JA, Wong MQ, Owen DA, et al. Metronomic gemcitabine suppresses tumour growth, improves perfusion, and reduces hypoxia in human pancreatic ductal adenocarcinoma. Br J Cancer. 2010; 103: 52-60.

62. Norden AD, Young GS, Setayesh K, Muzikansky A, Klufas R, Ross GL, et al. Bevacizumab for recurrent malignant gliomas: efficacy, toxicity, and patterns of recurrence. Neurology. 2008; 70: 779-787.

63. Norden AD, Drappatz J, Wen PY. Novel anti-angiogenic therapies for malignant gliomas. Lancet Neurol. 2008; 7: 1152-1160.

64. Swartz HM, Walczak T. Developing in vivo EPR oximetry for clinical use. Adv Exp Med Biol. 1998; 454: 243-252.

65. Williams BB, Khan N, Zaki B, Hartford A, Ernstoff MS, Swartz HM. Clinical electron paramagnetic resonance (EPR) oximetry using India ink. Adv Exp Med Biol. 2010; 662: 149-156.

66. Swartz HM, Khan N, Buckey J, Comi R, Gould L, Grinberg O, et al. Clinical applications of EPR: overview and perspectives. NMR Biomed. 2004; 17: 335-351.

67. Khan N, Williams BB, Swartz HM. Clinical Applications of In Vivo EPR: Rationale and Initial Results. Applied Magn Reson. 2006; 30: 185-99.

68. Khan N, Hou H, Hein P, Comi RJ, Buckey JC, Grinberg O, et al. Black magic and EPR oximetry: from lab to initial clinical trials. Adv Exp Med Biol. 2005; 566: 119-125.

69. Kaanders JH, Bussink J, van der Kogel AJ. ARCON: a novel biology-based approach in radiotherapy. Lancet Oncol. 2002; 3: 728-737.

70. Janssens GO, Rademakers SE, Terhaard CH, Doornaert PA, Bijl HP, van den Ende P, et al. Accelerated radiotherapy with carbogen and nicotinamide for laryngeal cancer: results of a phase III randomized trial. J Clin Oncol. 2012; 30: 1777-1783.


Tumor hypoxia (pO2 ; partial pressure of oxygen < 10 – 15 mmHg) plays a critical role in radio-resistance and promotes the development of aggressive tumor phenotypes. Furthermore, tumor hypoxia is dynamic and varies with tumor type, stage and as a consequence of ionizing radiation and other therapies. In spite of its profound effect on treatment outcome, tumor pO2 has been sub-optimally exploited in radiation oncology. Current radiotherapy plans do not take into account specific temporal changes in individual tumor pO2 levels due to lack of appropriate oximetry techniques, and therefore potentially may be suboptimal. In particular, hypofractionated treatments are increasingly used with large doses (4 - 20 Gy) of ionizing radiation that may have different effects on the levels of oxygen in individual tumors during the course of treatment. Real-time monitoring of tumor pO2 might make it feasible to improve the outcome by scheduling fractions at times of increased tumor pO2 . Such tumor oxygen guided treatment protocols can only be accomplished by oximetry techniques that can provide accurate serial measurements of tumor pO2 throughout the course of therapy.

We have pioneered in vivo EPR oximetry using micro-particulate oxygen-sensing probes for real-time monitoring of tissue pO2 in superficial (<10 mm) as well as deep-sited tumors, repeatedly and accurately, for clinical applications. Our goal is to improve treatment outcome by providing the information about dynamic tumor oxygen levels so that irradiations can be scheduled when the tumors are better oxygenated either with or without oxygen enhancing interventions e. g. pre-irradiation, carbogen (2 - 5% CO2 balance O2 ) inhalation, hyperthermia, and anti-angiogenic treatment. Temporal changes in tumor pO2 can also be used as a prognostic marker to predict efficacy, identify responders and non-responders, and individualize therapy. An overview of in vivo EPR oximetry, pre-clinical results and the current status of clinical oximetry are briefly described to highlight the potential advantages of EPR oximetry in radiation oncology.


Khan N, Hou H, Chen EY, Jarvis LA, Schaner PE, et al. (2013) Bench-To-Bedside Oximetry for Real-Time Monitoring of Tumor Po2 : A Critical Parameter Which Influences Radiotherapeutic Outcome. J Radiol Radiat Ther 1(3): 1017.


•    pO2;
•    Hypoxia;
•    Hyperoxia;
•    Carbogen;
•    Radiotherapy;
•    Oxygen sensors;
•    EPR oximetry


Radiotherapy is an important therapeutic modality for both curative and palliative cancer treatment with nearly 50% of cancer patients undergoing radiotherapy at some point during their illness. The level of oxygen in solid tumors is one of the key pathological parameters that can affect the outcome of treatment. It has been shown that radiosensitivity decreases by a factor of up to three when the pO2 declines from radiobiologically normoxic levels (>15 – 20 mmHg) to profound hypoxia [1,2]. Several animal and human tumor xenograft studies indicate that acute hypoxia due to transient cessations in microregional blood flow, in addition to chronic hypoxia arising from limitations in oxygen diffusion, may be a significant cause of radiation resistance [3,4]. Tumor hypoxia is also implicated in other aspects of tumor development including genomic instability, aggressive phenotypes, metastases, and loss of apoptosis [3,5-7]. In some cases, one-time measurement of tumor pO2 prior to treatment can be a good predictor of the outcome. A poor prognosis of patients with pre-treatment tumor pO2 of less than 10 mmHg has been reported for tumors of various origins, including soft tissue sarcoma, head and neck cancer, cervical cancer and esophageal cancer [8-12]. The single assessment of tumor pO2 in these studies was carried out using invasive electrode techniques or indirect markers of tissue hypoxia (e. g. pimonidazole staining). However, a single measurement cannot provide the crucial temporal information necessary to guide the treatment as the tumor pO2 will vary in a complex time-dependent manner during the course of radiotherapy.

The temporal changes in tumor pO2 can also be induced by radiation through a combination of several factors such as, cell killing, direct vascular damage, and change in interstitial fluid pressure [13,14]. In experimental tumors, an increase or decrease in tumor pO2 after single small or large doses of radiation has been observed [15-17]. Consequently, it is very desirable to be able to measure tumor pO2 accurately and repeatedly over the course of radiotherapy. Such information will be extremely useful to (i) evaluate and optimize the effectiveness of methods being developed to increase tumor pO2 such as carbogen inhalation and anti-angiogenic therapies, and (ii) improve treatment outcomes by reducing the levels of hypoxia and scheduling irradiations when the tumors are well oxygenated. The measurements can be achieved using in vivo EPR oximetry, which provides repeated measurement of absolute tumor pO2 at one or more sites simultaneously [18-20]. We have successfully implemented in vivo EPR oximetry using small (microcrystalline) probes for discrete measurement of tumor pO2 at one or more sites to investigate the effect of radiotherapy and hyperoxic adjuvant approaches on tumor pO2 with a goal to optimize treatment outcome [15,21-26].

In vivo EPR oximetry for repeated measurement of tumor pO2

The basis of EPR oximetry is the paramagnetic nature of molecular oxygen (O2 ), which therefore affects the EPR spectra of other paramagnetic materials in its vicinity by altering their relaxation rates [18-20,27]. The magnitude of this relaxation effect is directly related to the amount of oxygen that is present in the environment of the paramagnetic materials (oximetry probes). Accordingly, the line width of the EPR spectrum of an oximetry probe, when injected into a tissue of interest, provides a direct measurement of tissue pO2 . Several oximetry probes have been developed with varying sensitivities to oxygen, including the crystals of lithium phthalocyanine (LiPc) and its analogs [28-30]. These crystals are metabolically inert and very sensitive for measuring low levels of oxygen, which makes them especially suitable for pO2 measurements in tumors [27,28,31]. For clinical applications, these oximetry probes have been encapsulated in oxygen-permeable biocompatible and inert polydimethylsiloxane (PDMS) polymer or Teflon AF2400 [31- 35]. In vivo EPR oximetry requires a one-time implantation of the oximetry probe (minimally invasive procedure using 25- 23 gauge needles or during surgery for other procedures) but all subsequent procedures for pO2 measurement are entirely noninvasive and can be repeated as often as desired (from few seconds to several weeks). A surface-loop resonator is positioned over the tissue implanted with the oximetry probe and by the use of an appropriate combination of an exciting frequency (1200 MHz for L-band) and a magnetic field (400 Gauss) [36]. The scanning of the magnetic field produces a characteristic EPR signal. By using an appropriate calibration of the oximetry probe used, the line width of the EPR signal provides a sensitive measurement of tissue pO2 . In order to assess the heterogeneity in pO2 , oximetry probes can be implanted at different sites within a tumor and a simultaneous measurement of pO2 from each implant can be carried out using magnetic field gradients (multi-site EPR oximetry) [37-39].

In vivo EPR oximetry offers several unique capabilities and advantages compared to other approaches:

(i) A direct measurement of actual oxygen content in a tissue of interest, whereas indirect techniques such as BOLD (blood oxygen-level dependent) MRI and NIR (near-infra-red) derive oxygen information from hemoglobin saturation in the vasculature.

(ii) The measurements provide quantitative pO2 data (techniques such as misonidazole or EF5 provide information on the occurrence of hypoxia but usually do not provide quantitative pO2 information).

(iii) The measurements can be made continuously and repeatedly as desired, without a confounding influence of prior measurements or a reduction in sensitivity.

(iv) The oximetry probes are metabolically inert and therefore do not perturb the tissue microenvironment including oxygen content (electrochemical techniques such as Eppendorf are invasive and require the consumption of oxygen for pO2 assessment).

(v) Tumor pO2 measurements can be made up to a depth of 10 mm from the surface using direct implantation and detection of the particulate oximetry probes.

(vi) Implantable oxygen sensors (ImOS) can be used for pO2 measurement at depths ranging from 3 mm to greater than 20 cm with excellent detection sensitivity. The particulate oximetry probes are loaded on the sensory tip of the implantable resonators.

(vii) Particulate oximetry probes are deposited via a 23 - 25 gauge needle. ImOS can be implanted via a similar catheter and incision to position the subcutaneous coupling loop, or carried out in conjunction with a scheduled surgery. All subsequent measurements are entirely noninvasive.

(viii) There is no other technique available at present to make repeated measurement of absolute tumor pO2 without the need to reintroduce the probe for each measurement.

We have extensively used EPR oximetry to study the temporal variations in tissue pO2 in a wide range of pathologies including experimental and human xenograft tumors, muscle, heart, brain, kidney, and liver in animal models [15,18,21-26,33,40- 50]. Recent preclinical and clinical oximetry results are briefly described below.

Experimental and human xenograft tumor pO2 during radiotherapy and/or hyperoxic challenge

Using in vivo EPR oximetry, we have serially measured ectopic and orthotopic tumor pO2 and the results indicate that

(i) The pre-treatment (baseline) pO2 varies with the tumor type and size [15,21,24,26,51].

(ii) The extent and timing of post-irradiation changes in pO2 depends on the tumor type and radiation dose [15,21,25].

(iii) The outcome of radiotherapy can be enhanced if the tumors are irradiated at the time of an increase in tumor pO2 compared to when they are hypoxic [23,25].

(iv) A significant increase in tumor pO2 may be observed when inhaling carbogen (5% CO2 balance O2 ). However, not all the tumors response to carbogen (inter and intra-tumor variability) and also the magnitude of increase in pO2 usually declines with tumor growth over days [15,21,24,25].

(v) Anti-angiogenic approaches such as metronomic chemotherapy [52-54] can significantly increase tumor pO2 . Therefore, a serial measurement of pO2 is necessary to identify therapeutic window during which the tumors are oxygenated for effectual combination with radiotherapy [46,51,55]. However, the efficacy of such treatment varies with the tumor type and the chemotherapeutic agent (unpublished results).

We are currently using multiple site ImOS to simultaneously measure orthotopic human xenograft glioma and contralateral brain pO2 during hyperoxia with carbogen inhalation and metronomic chemotherapy using gemcitabine. The tissue pO2 of the contralateral brain reflects the global changes in pO2 , allowing us to differentiate between those and glioma specific changes in pO2 . The ImOS are assembled from enameled copper wires (thickness: 0.15 mm) and consists of a coupling loop (8 - 10 mm diameter) and transmission lines (3-mm length, i. e. depth from the surface, but capable of being any length) with sensory loops (or tips, 0.2 - 0.3 mm diameter) loaded with a few LiPc crystals [22,24,35,56], Figure 1. The entire assembly is then coated with oxygen-permeable and biocompatible Teflon AF2400 [35]. For intracranial pO2 measurements, the transmission lines with sensory loops are gently inserted into the brain tissue while the coupling loop remains on the top of the skull under the skin. The coupling loop is inductively coupled with an external surface loop resonator of L-band EPR spectrometer for pO2 measurements. ImOS with single or multiple sensory loops and varying length of the transmission lines are shown in Figure 1D. Such ImOS designs are suitable for single site or multiple site measurements in deep sited tumors, e. g. head and neck or prostate cancer.

We are currently pursuing an Investigational Device Exemption (IDE) from the FDA for clinical use of ImOS for pO2 measurements. The mean area at the surface of each sensory loop is estimated to be 0.03 - 0.07 mm2 . EPR oximetry therefore samples a region that includes several capillaries and a region that spans the local heterogeneous tumor structure [18,24,57].

Tumor pO2 measurement at more than one site using multi-site EPR oximetry is expected to resolve any gradient in pO2 that may exist across a tumor. The EPR spectrum acquired by a twoprobe (sensory loop) ImOS in a mouse brain using multisite EPR oximetry is shown in Figure 2A. The temporal changes in glioma and contralateral brain pO2 measured simultaneously before and during carbogen inhalation are shown in Figure 2B. First, a baseline pO2 was measured in the anesthetized mouse breathing 30% O2 (30 min) and then the mouse was allowed to inhale carbogen (25 min) and the breathing gas was again switched back to 30% O2 for 15 min.

The mean (±SD) baseline pO2 of the contralateral brain and U251 glioma were 66.0±2.9 and 15.0±0.8 mmHg respectively, which significantly increased during carbogen breathing. In these experiments, the baseline glioma pO2 and response to carbogen varied among the animals. The typical changes in the contralateral brain and U251 glioma pO2 in two animals measured repeatedly for 5 consecutive days are shown in Figure 3. A significant increase in contralateral brain pO2 was evident in both animals. However, the extent of the increase in pO2 was different between the animals and also varied over days. A minimal change in the pO2 of the glioma was observed in one of the animals with carbogen inhalation (Figure 3A). On the other hand, a significant increase in the glioma pO2 occurred in another animal, but the response to carbogen quickly declined by day 3 with no change in pO2 on days 4 and 5. These results indicate a considerable variation in response to carbogen inhalation between the animals as well as over days in the same animal. Consequently, a real-time monitoring of tumor pO2 is necessary to assess day to day variation in the response to carbogen inhalation. This information will be especially useful to identify the tumors that are oxygenated and therefore will be sensitive to radiotherapy.

Several anti-angiogenic approaches, such as bevacizumab or metronomic chemotherapy, are being investigated to normalize the chaotic and inefficient vasculature to restore blood flow and improve drug delivery in the tumors [52-54,58]. However, in order for these approaches to be successful, it is desirable to directly measure their effect on tumor vasculature or identify surrogate markers that can be repeatedly monitored during the treatment. We anticipate that the restoration of blood flow into the tumors should increase pO2 , and therefore if measured repeatedly, can be used as a marker to assess the effectiveness of anti-angiogenic treatments. To test this hypothesis, we are currently investigating the effect of metronomic chemotherapy with gemcitabine on the pO2 of human xenograft U251 glioma, Figure 4. The dose and schedule of gemcitabine in this study were derived from the various metronomic treatment reported in preclinical studies [59-61]. In these experiments, the untreated U251 gliomas were hypoxic with a mean pO2 of 6 - 9 mmHg on day 1. No significant change in the pO2 of the control group was observed during glioma growth for five consecutive days. A significant increase in the glioma pO2 only on day 3 was evident in the mice treated with a single dose of 150 mg/kg gemcitabine on day 1. On the other hand, glioma pO2 increased significantly from day 3 - day 5 when the mice were treated with metronomic gemcitabine (30 mg/kg x 5 and 10 mg/kg x 4). Such increases in glioma pO2 are expected to significantly improve treatment outcome when combined with radiotherapy. However, the profile of temporal changes in glioma pO2 varied with different gemcitabine dose/ schedule, which highlights the need for repeated measurements of glioma pO2 for efficacious combination of such approaches with radiotherapy.

Bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), has been approved by FDA as a single agent to treat glioma patients with progressive disease after prior therapy. This antibody has shown antiangiogenic effect with a transient normalization of vasculature in the gliomas [54,62,63]. However, lack of methods to repeatedly measure and follow vessel normalization has been a rate limiting step in rationally translating these approaches into the clinic and in developing efficacious protocols for combination therapies. A real-time monitoring of tumor pO2 during hyperoxic or antiangiogenic approaches will provide critical information on tumor oxygenation and/or vessel normalization that can be used to predict outcome, identify non-responders and efficiently combine these approaches with radiotherapy to improve therapeutic outcome.

Current status of clinical EPR oximetry

We have developed an L-band (1.2 GHz) EPR spectrometer using a permanent magnet with a gap of 50 cm between the poles of the magnet to comfortably position most human subjects for oximetry measurements [36,64]. The suitability of the clinical EPR system for pO2 measurements in superficial tumors (< 10 mm depth) using India ink as an oximetry probe has been demonstrated for locations ranging from the feet, to the anterior and posterior surfaces of the torso, and scalp [18,64-68]. To date, we have performed repeated pO2 measurements in superficial tumors at different sites of 14 patients. The tumors varied considerably in their baseline pO2 (0 to 10 mmHg). Furthermore, the response to breathing enriched oxygen varied considerably between the patients with a significant increase in pO2 in some tumors while others had minimal or no change in pO2 from the baseline. These results demonstrate the feasibility of in vivo EPR oximetry in the clinical setting to make repeated noninvasive direct measurements of tumor pO2 . Importantly, variability in baseline tumor pO2 and response to enriched oxygen signify the need for a direct measurements of pO2 during hyperoxic approaches to confirm, if any, increase in pO2 for effective combination with radiotherapy.


The extent of the influence of hypoxia in tumorigenesis makes it a critical factor that must be targeted to achieve therapeutic benefit. The availability of oximetry methods that can provide real-time monitoring of tumor pO2 are key for improving the outcome of radiotherapy by refining the standard protocols (optimize the timing of combined therapy involving radiation and hyperoxic/anti-angiogenic approaches) and by providing patientspecific pO2 information to individualize therapy (by altering the timing of fractions to maximize therapeutic effect for a specific patient). Extensive preclinical and initial clinical studies have demonstrated the potential of EPR oximetry to fulfill this need. An expected application of clinical EPR oximetry will be in the ARCON trials [69,70]. In these trials, carbogen and nicotinamide is being used to reduce hypoxia, however, no direct measurement of tumor pO2 was made to confirm whether the tumors indeed have or have not been oxygenated. Direct pO2 measurements with in vivo EPR oximetry will allow the identification of patients who have responded to carbogen breathing and therefore will benefit from radiotherapy. The results can also be used to evaluate the outcomes between the cohorts who did or did not have an increase in tumor pO2 to correctly validate the trials. In concluding, we anticipate that in vivo EPR oximetry is likely to play a crucial role in radiation oncology for prognosis and identify responders to individualize therapy.


Pilot Program Project funded by the Norris Cotton Cancer Center, Department of Radiology, and the EPR Center, Geisel School of Medicine at Dartmouth, Hanover, NH.

Received : 09 Sep 2013
Accepted : 14 Nov 2013
Published : 19 Nov 2013
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