Christopher G. Milross

Interlaboratory variation in oxygen tension measurement by Eppendorf “Histograph” and comparison with hypoxic marker

The median of pO2 values in tumor measured by Eppendorf “Histograph” with a needle‐type electrode has been used as a prognostic indicator in cancer patients. However, it is not established that a pretreatment measured pO2 value can be used as a universal predictor of local control probability, because the variation in pO2 values, especially in hypoxic tissue, among institutes may not allow comparison of measured “absolute pO2 values.” The purpose of this study was to examine the variation in oxygen tension measurement by Eppendorf “Histograph” among six laboratories using a single batch of mice and tumors and the same detailed protocol. These results were also compared to the immunohistochemical staining of 2‐nitroimidazole adducts.

Tumor reoxygenation as a mechanism of taxol-induced enhancement of tumor radioresponse

Paclitaxel is a novel chemotherapeutic agent that arrests cells in the radiosensitive G2 and M phases of the cell cycle and as such may act as a specific cell cycle radiosensitizer. We recently reported that paclitexel induces mitotic arrest in the MCA-4 murine mammary carcinoma and enhances radio-response of this tumor. However, the greatest enhancement was observed not when radiation was given at the time of peak mitotic arrest, which was 9 h after paclitaxel administration, but when it was given 24 h after paclitaxel. This implied the involvement of other mechanisms in radiosensitization; we hypothesized that tumor reoxygenation was a likely mechanism based on the observed massive loss of mitotically arrested cells at 24 h. The present study shows that paclitaxel greatly enhanced MCA-4 tumor radioresponse when radiation was given under air-breathing conditions (DMF = 1.74), but not when it was performed under hypoxic conditions. This observation supports the hypothesis of tumor reoxygenation as a mechanism of enhancement of tumor radioresponse. That reoxygenation occurred in tumors treated with paclitaxel 24 h earlier was confirmed by direct measurements of pO2 values, using the Eppendorf pO2 histograph. Median pO2 values increased from 6.2 mmHg in untreated tumors to 10.0 mmHg in tumors treated with paclitaxel. These observations emphasize the importance of timing of paclitaxel administration in relation to radiation treatment.

Enhanced radioresponse of paclitaxel-sensitive and -resistant tumours in vivo

Paclitaxel is a potent chemotherapeutic drug and also has the potential to act as a radioenhancing agent. The latter is based on its ability to arrest cells in the radiosensitive G2M phases of the cell cycle; the weight of supporting evidence is derived mainly from in vitro studies. Our previous in vivo experiments identified enhanced tumour radioresponse predominantly attributable to tumour reoxygenation occurring as a result of paclitaxel-induced apoptosis. The current study investigated whether paclitaxel enhanced the radioresponse of tumours which are insensitive to apoptosis induction, but exhibited mitotic arrest, and compared the degree and kinetics of the response to that in tumours which develop apoptosis. The mouse mammary carcinoma MCa-29 (apoptosis sensitive) and the squamous cell carcinoma SCC-VII (apoptosis resistant) were used. In addition, the study investigated whether paclitaxel affected normal skin radioresponse to determine if a therapeutic gain could be achieved. Paclitaxel enhanced the radioresponse of both types of tumours. In the SCC-VII tumour, radiopotentiation occurred within 12 h of paclitaxel administration coincident with mitotic arrest, where enhancement factors (EFs) ranged from 1.15 to 1.37. In MCa-29 tumour, the effect was greater, EFs ranging from 1.59 to 1.91 and occurred between 24 and 72 h after paclitaxel when apoptosis was the predominant microscopic feature of treated tumours and when tumour oxygenation was found to be increased. The acute skin radioresponse and late leg contracture response were essentially unaffected by prior treatment with paclitaxel. Therefore, by two distinct mechanisms, paclitaxel was able to enhance the radioresponse of paclitaxel-sensitive and -resistant tumours, but not the normal tissue radioresponse, thus providing true therapeutic gain.

The effect of tumor size on necrosis and polarographically measured pO2

Tumor necrosis and oxygen status were investigated as a function of tumor size in three syngeneic murine carcinomas, MCa-4, OCa-I, and SCC-VII, in C3Hf/Kam mice. Tumor necrosis was estimated histologically, and tumor oxygenation determined by direct polarographic histography. As tumor volume increased necrosis increased significantly in all three tumor types (p < 0.001). Similarly, as tumor volume increased from 200 to 1400 mm3, hypoxia, defined as the percentage of measured pO2 values < or = 5.0 mm Hg, increased from 55.1% to 95.9%, 70.3% to 81.4%, and 56.8% to 98.5% in MCa-4, OCa-I, and SCC-VII tumors respectively (p < 0.001). Correcting pO2 for necrosis reduced the tumor size dependence of measured tumor hypoxia in all three tumor types but in no case was the reduction significant. The main effect of correction was to shift the fitted curves of percent pO2 values < or = 5.0 mm Hg down toward lower percentages for all tumors. This change was significant for MCa-4 and OCa-1 tumors (p < 0.001), but not for SCC-VII (p = 0.054). Defining the influence of variables such as necrosis that affect polarographic assessment of tumor oxygenation is important to enhance the techniques reliability and prospect as an investigative and predictive tool.

Therapeutic potential of paclitaxel-radiation treatment of a murine ovarian carcinoma

BACKGROUND Paclitaxel has been shown to radiosensitize tumor cells in culture by arresting them in the most radiosensitive G2 and M cell cycle phases. In vivo preclinical studies are now necessary to obtain full insight into the radiopotentiating potential of this drug and its ability to increase the therapeutic gain of radiotherapy. We tested its ability to enhance the tumor radioresponse of an ovarian carcinoma and to influence the normal tissue radioresponse of recipient mice. METHODS Mice bearing 8-mm isotransplants of a syngeneic ovarian carcinoma, designated OCA-I, in their legs were treated with 40 mg/kg paclitaxel i.v., 14-60 Gy single-dose local tumor irradiation, or both; radiation was given under ambient conditions 1-96 h after paclitaxel. Tumor growth delay, tumor cure rate (TCD50 assay), and delay in tumor recurrences were measured. Normal tissue radioresponse was determined using jejunal crypt cell survival at 3.5 days after exposure of mice to 9-14 Gy single dose of total body irradiation; the mice were untreated or treated with 40 mg/kg i.v. paclitaxel 4-96 h before irradiation. RESULTS Paclitaxel alone was effective against OCA-I, but its combination with irradiation produced supra-additive tumor growth delay. It also reduced TCD50 values and delayed tumor recurrences. The enhancement of tumor radioresponse ranged from 1.33 to 1.96; the value increased as the time between paclitaxel administration and tumor irradiation increased up to 48 h, but then decreased again at 96 h. In contrast, paclitaxel protected jejunum against radiation damage by factors of 1.03 to 1.07 when given 24-96 h before irradiation. It showed some potentiation of damage (by a factor of 1.07), but only when given 4 h before irradiation. CONCLUSIONS Paclitaxel potentiated tumor radioresponse if given within 4 days before irradiation, whereas it caused radioprotection of normal tissue (jejunum) at that time. Therefore, paclitaxel significantly increased therapeutic gain and so has potential for use in combination with radiotherapy for pelvic malignancies.

The effect of paclitaxel on the cell cycle kinetics of a murine mammary adenocarcinoma in vivo

Abstract: This article reports data that describe perturbations in cell‐cycle dynamic kinetic parameters following in vivo treatment of a syngeneic murine mammary tumor (MCa‐4) with paclitaxel. The kinetic parameters include the duration of the S (Ts) and G2M (TG2M) phases of the cell cycle, the tumor potential doubling time (Tpot), the bromodeoxyuridine (BrdUrd) labeling index (Ll), and the fraction of proliferating cells that successfully completed mitosis following paclitaxel treatment. When tumors transplanted into the right thigh of 4‐month‐old male C3Hf/kam mice grew to 8 mm mean diameter, the mice were injected intravenously with 40 mg/kg paclitaxel. At one of a range of times thereafter (1–72 hours) animals were pulse labeled with 60 mg/kg BrdUrd. Mice were sacrificed and tumors excised either immediately or at 3 or 6 hours after BrdUrd labeling. Single‐nuclei suspensions were prepared, and the kinetic parameters evaluated using bivariate DNA versus BrdUrd flow‐cytometric methods.

Evaluation of [131I]iodoerythronitroimidazole as a predictor for the radiosensitizing effect

The aim of this study was to evaluate whether radiolabeled iodoerythronitroimidazole (IETNIM) could predict the radiosensitization effect on tumors. Tumor-bearing mice were irradiated at a dose of 25, 31 and 37 Gy after the injection of IETNIM. They were also exposed to 37 Gy radiation at 35, 70, 140 and 240 min after the i.p. injection of IETNIM. After the irradiation, tumor growth assays were conducted and the effect of IETNIM as a radiosensitizer was estimated as enhancement factor (EF). Tumor uptake was measured at 35, 70, 140 and 240 min after i.p. injection of [131I]IETNIM, which were the same intervals used in the radiosensitization study. EF of IETNIM in mice treated with 25, 30 and 37 Gy irradiation was 0.72, 0.98 and 1.28, respectively. EF of IETNIM in mice irradiated at 35, 70, 140 and 240 min after the injection was 1.50, 1.69, 1.46 and 1.08, which corresponded to the tumor uptake and blood clearance of [131I]IETNIM. [131I]IETNIM may be a suitable radiopharmaceutical to predict the radiosensitization effect of misonidazole analogs on tumors.