Conclusions: Our results suggest that both HIF-1 dependent and independent pathways are involved in the downregulation ofVEGF expression by gefitinib. Gefitinib also appears to increase tumor oxygenation, which may have important implicationsfor the use of this drug in conjunction with radiotherapy.

227 Observation of Increase of Oxygen in Experimental Murine Tumors Approximately 20 Minutes After X-Ray Irradiation

H. Hou, N. Khan, H. Yu, E. Demidenko, J. O’Hara, H.M. Swartz

EPR Center for the Study of Viable Systems, Hanover, NH

Purpose/Objective: The development of in vivo EPR oximetry now makes it feasible to follow the time course of oxygenationin tumors non-invasively, continuously, and repeatedly. In principle this should be very valuable because radiation response ofhypoxic tumors is very sensitive to the level of oxygen and tumor growth, radiation therapy, and chemotherapy all potentiallycan change tumor pO2 dynamically. Using EPR continuously to follow early post-irradiation changes in RIF-1 tumors receivingfractionated radiation, we observed a previously unobserved transient increase in tumor oxygenation during the first hour.

Materials/Methods: Two aggregates of oxygen-sensitive lithium phthalocyanine (LiPc) were implanted in 200–600 mm3RIF-1 tumors in 15 C3H/HEJ mice (11 irradiation and 4 sham control). Three days after implantation the tumors received 4Gy x-rays at 3.1 Gy/min. Continuous measurements of tumor pO2 were made before and for 70 minutes after irradiation forfive consecutive days.

Results: The baseline (and change) in tumor pO2 values for the five days were: day 1: 5.5 (�1.7), day 2: 5.0 (�2.1), day 3:5.0 (�1.9), day 4: 6.1 (�1.4), day 5: 5.3 (�6.4) torr. All changes from baseline were statistically significant, as was the increaseon day 5 compared to days 1–4 (p�0.01 for all comparisons). The maximum increases were at 15.6–22.5 minutes postirradiation and returned to baseline at 25.7-35.2 minutes.

Conclusions: The unique capabilities of in vivo EPR oximetry enabled us to follow in detail the kinetics of tumor pO2post-irradiation. The occurrence and magnitude of the observed post-irradiation increase provides a potentially powerfulwindow for enhancing the therapeutic ratio, by delivering a second dose during this rebound period in which the biologicaleffects on the tumor should be enhanced significantly. These results also indicate the potential value of methods for detailedmonitoring of tumor pO2 after irradiation.

228 Radiation, Hypoxia and Chemotherapy-mediated Gene Therapy and the Potential for Tumour Delivery viaMacrophages

S.D. Scott,1 O. Greco,2 C. Murdoch,1 C. Lewis1

1Division of Geonomic Medicine, Fairfax, United Kingdom, 2Division of Clinical Sciences, Sheffield, S. York, UnitedKingdom

Purpose/Objective: Hypoxia is an important prognostic indicator of poor tumour treatment outcome after radio- andchemo-therapy. We have developed suicide gene therapy vectors containing radiation-responsive CArG elements and hypoxia-responsive elements (HREs), which are activated by clinical levels of ionizing radiation and hypoxia. We have successfullyshown vector responses to both fractionated X-ray doses of 1–5 Gy and 0–1% O2. Furthermore, we are testing the use of humanmacrophages for delivery of gene therapy to tumours. Macrophages accumulate preferentially in malignant tissue, in particular,necrotic/hypoxic areas.

Materials/Methods: We first examined the possibility of vector activation via neutron irradiation and chemotherapeutic agents.Human MCF-7 breast adenocarcinoma cells were transfected with vectors in which CArG promoters controlled expression ofthe herpes simplex virus thymidine kinase (HSVtk) suicide gene. Neutron irradiation (1.3 Gy) produced at least as highvector-mediated tumour cell kill as 2 Gy of X-rays. Likewise, clinically-relevant doses of cisplatin and doxorubicin were ableto drive effective HSVtk-therapy via CArG promoters. Secondly, we have established an in vitro human T47D breast tumourcell spheroid model. These spheroids developed necrotic/hypoxic centres as they grew, mimicking a characteristic feature ofsolid tumours. Macrophages derived from fresh blood monocytes were labeled and co-cultured with the spheroids, which werethen analysed for macrophage infiltration via immunostaining and flow cytometry.

Results: Results revealed extensive macrophage infiltration of spheroids, with predominance in the peri-necrotic, hypoxicregions. We are now conducting studies using vector-transduced macrophages, and will analyse hypoxia-regulated transgeneexpression following spheroid infiltration.

Conclusions: In conclusion, we have produced gene therapy vectors responsive to clinical doses of hypoxia, X- andneutron-irradiation and common chemotherapeutic agents. We have also shown that human macrophages infiltrate a tumourspheroid model, as observed in tumours in situ. The combining of the vector and delivery technologies represents a verypromising direction for targeted anticancer gene therapy.

229 Tumor Hypoxia

A. Koong

The Center for Advance Medicine, Clinical Cancer Center,, Stanford University, Stanford, CA

Purpose/Objective: Hypoxia within solid tumors decreases therapeutic efficacy and identification of hypoxia markers mayinfluence the choice of therapeutic modality.

S137Proceedings of the 47th Annual ASTRO Meeting