Radiosensitization of murine tumors by Fluosol-DA 20%

The effect of perfluorochemicals in combination with carbogen breathing on the response of SCK tumors of mice to fractionated irradiation was investigated. The SCK tumors of A/J mice were irradiated twice a day at 3 Gy per fraction (6 Gy per day), with a total dose of 18 Gy over 3 days. When the host animals were treated with an intravenous (iv) injection of 12 ml/kg of Fluosol-DA 20% before the first daily tumor irradiation and carbogen breathing during every X irradiation with Fluosol-DA 20% injection without carbogen breathing. The hypoxic cell fraction, as determined by an in vivo-in vitro cloning assay, decreased significantly, and the intratumor pO2, as determined with microelectrodes, was markedly increased by Fluosol-DA 20% injection and carbogen breathing. It was concluded that oxygenation of hypoxic cells in SCK tumors during the course of fractionated irradiation was improved by the iv injection of Fluosol-DA 20% and carbogen breathing.     

Measurements of tumor tissue oxygen tension using a time-resolved luminescence-based optical oxylite probe: comparison with a paired survival assay

Recently, a system that measures tissue oxygen tension using time-resolved luminescence-based optical sensors has become available commercially (Oxford Optronix, Oxford, England). Two experiments were conducted using this system. First, the oxygen tension distribution was measured in two tumor lines: a spontaneous mouse fibrosarcoma, FSa-II, and a human squamous cell carcinoma xenograft, FaDu. The area in which the pO(2) was equal to or lower than 2.5 mmHg was defined as the hypoxic lesion, and the hypoxic cell fraction was taken as the fraction of these measurements in a tumor. The measured hypoxic cell fractions were compared with those determined by the paired cell survival assay for tumors of various sizes. Second, the tumor tissue pO(2) was measured continuously after administration of two different anesthetics to evaluate the effect of these drugs on tissue pO(2). Results indicated a good agreement between the hypoxic cell fractions measured by this system and those determined by the paired cell survival curve assay for tumors smaller than approximately 500 mm(3). For tumors larger than approximately 500 mm(3), the hypoxic cell fractions measured by the oxygen probe system were higher than those measured by the paired cell survival assay. This may suggest that the hypoxic cell fraction measured by the oxygen probes included both hypoxic and necrotic areas in large tumors where necrotic lesions occupied a significant portion of the tumor. Continuous measurements of pO(2) after anesthesia (Nembutal, or ketamine plus xylazine) showed a consistent rise in the pO(2) during the first 20-30 min of measurement. Subsequently, the pO(2) values became constant or continued to rise slowly. For comparison, the tumor cell survivals were assayed after a dose of 20 Gy given in air at 5, 20 and 60 min after anesthesia. The result showed a decrease in cell survival only in tumors irradiated 20 min after an injection of Nembutal.     

Radiation-induced IFN-gamma production within the tumor microenvironment influences antitumor immunity

Alterations to the tumor microenvironment following localized irradiation may influence the effectiveness of subsequent immunotherapy. The objective of this study was to determine how IFN-gamma influences the inflammatory response within this dynamic environment following radiotherapy. B16/OVA melanoma cells were implanted into C57BL/6 (wild-type (WT)) and IFN-gamma-deficient (IFN-gamma-/-) mice. Seven days after implantation, mice received 15 Gy of localized tumor irradiation and were assessed 7 days later. Irradiation up-regulated the expression of VCAM-1 on the vasculature of tumors grown in WT but not in IFN-gamma-/- mice. Levels of the IFN-gamma-inducible chemokines MIG and IFN-gamma-inducible protein 10 were decreased in irradiated tumors from IFN-gamma-/- mice compared with WT. In addition to inducing molecular cues necessary for T cell infiltration, surface MHC class I expression is also up-regulated in response to IFN-gamma produced after irradiation. The role of IFN-gamma signaling in tumor cells on class I expression was tested using B16/OVA cells engineered to overexpress a dominant negative mutant IFN-gamma receptor (B16/OVA/DNM). Following implantation and treatment, expression of surface class I on tumor cells in vivo was increased in B16/OVA, but not in B16/OVA/DNM tumors, suggesting IFN-gamma acts directly on tumor cells to induce class I up-regulation. These increases in MHC class I expression correlated with greater levels of activated STAT1. Thus, IFN-gamma is instrumental in creating a tumor microenvironment conducive for T cell infiltration and tumor cell target recognition.     

Regional blood-to-tissue transport in an irradiated rat glioma model

To assess vascular permeability in intracerebral grafts of the 36B-10, F-344 rat glioma following 20 Gy 137Cs whole brain irradiation, the blood-to-tissue transport constant, K, of [14C]-alpha-aminoisobutyric acid (AIB) was measured with quantitative autoradiography. Mean, 90th percentile, and 95th percentile values of K were determined in individual tumors and in treatment groups. In 15-day-old unirradiated control tumors, mean, 90th percentile, and 95th percentile values of K were, respectively, 11.3, 18.4, and 20.8 ml kg-1 min-1. In 15-day-old tumors irradiated on Day 14 (Day 1 postirradiation tumors) the K values were 5.9, 9.4, and 10.4, all of which were significantly less than the respective control values (P less than 0.01). In 16-day-old tumors irradiated on Day 14 (Day 2 postirradiation tumors), the K values were 10.8, 15.0, and 16.0, respectively, none of which was significantly different from control tumors. Mean K values for Day 2 vs Day 1 postirradiation tumors (10.8 vs 5.9) yielded P less than 0.05, but the 90th percentile and 95th percentile values for Day 2 vs Day 1 yielded 0.05 less than P less than 0.10. Separate experiments measured AIB and 86RbCl uptake in 36B-10 cells in vitro 1 and 2 days following 20 Gy irradiation to assess whether this radiation dose reduced the capacity of tumor cells to trap AIB or Rb+. Irradiation did not reduce the accumulation of either tracer, but rather was associated with an increased accumulation of AIB. Therefore, the AIB transport data suggest that vascular permeability and/or surface area decreases significantly in the day following 20 Gy irradiation and that this decrease reverses by the second day following irradiation.     

Isolation and characterization of highly radioresistant malignant hamster fibroblasts that survive acute gamma irradiation with 20 Gy

To study the acquired radioresistance of tumor cells, a model system of two cell lines, Djungarian hamster fibroblasts (DH-TK-) and their radioresistant progeny, was established. The progeny of irradiated cells were isolated by treating the parental cell monolayer with a single dose of 20 Gy (PIC-20). The genetic and morphological features, clonogenic ability, radiosensitivity, cell growth kinetics, ability to grow in methylcellulose, and tumorigenicity of these cell lines were compared. The plating efficiency of PIC-20 cells exceeded that of DH-TK- cells. The progeny of irradiated cells were more radioresistant than parental cells. The average D0 for PIC-20 cells was 7.4 +/- 0.2 Gy, which is three times higher than that for parental cells (2.5 +/- 0.1 Gy). Progeny cell survival in methylcellulose after irradiation with a dose of 10 Gy was 15 times higher than that of DH-TK- cells. In contrast to parental cells, the progeny of irradiated cells showed fast and effective repopulation after irradiation with doses of 12.5 and 15 Gy. The tumor formation ability of irradiated progeny cells was higher than that of parental cells; after 15 Gy irradiation, PIC-20 cells produced tumors as large as unirradiated progeny of irradiated cells, whereas the tumor development of DH-TK- cells diminished by 70%. High radioresistance of progeny of irradiated cells was reproduced during the long period of cultivation (more than 80 passages). The stability of the radioresistant phenotype of PIC-20 cells allows us to investigate the possible mechanisms of acquired tumor radioresistance.     

Inhibitory effects of WR-2721 and cysteamine on tumor initiation in mammary glands of pregnant rats by radiation

We evaluated the effect of WR-2721 [S-2-(3-aminopropylamino)-ethylphosphorothioic acid] and cysteamine (2-mercaptoethylamine) on the development of radiation-induced mammary tumors in rats. Pregnant rats were treated with WR-2721 or cysteamine 30 min prior to whole-body irradiation with gamma rays from a (60)Co source at a dose of 1.5 or 2.6 Gy. Additional pregnant rats were given saline and then exposed to gamma rays at a dose of 0, 1.5 or 2.6 Gy as a control. All rats were implanted with pellets of diethylstilbestrol, a tumor promoter, 1 month after termination of nursing and were observed for 1 year to detect palpable mammary tumors. No mammary tumors developed in the saline-injected nonirradiated rats. However, when rats were irradiated with 1.5 or 2. 6 Gy after saline treatment, the incidence of mammary tumors was high (71.4 and 92.3%, respectively). Administration of WR-2721 or cysteamine prior to irradiation with 1.5 Gy significantly decreased the tumor incidence (23.8 and 20.8%, respectively). Tumor prevention by either agent was less effective at the higher dose. The appearance of the first mammary tumor occurred later in rats treated with WR-2721 or cysteamine than in the control rats. An increasing rate of adenocarcinoma in the control group was observed with increasing dose from 1.5 Gy up to 2.6 Gy. However, the development of adenocarcinoma did not increase after pretreatment with WR-2721 or cysteamine in rats irradiated with 2.6 Gy. Many of the mammary tumors that developed in the control rats were of the ER(+)PgR(+) type. Administration of WR-2721 produced no tumors of the ER(+)PgR(+) type. Cysteamine treatment increased the development of ER-negative tumors. The serum concentration of progesterone was significantly higher in rats treated with WR-2721 or cysteamine than in the control rats. On the other hand, the estradiol-17beta concentration was reduced by treatment with WR-2721, but not significantly compared to the control. WR-2721 and cysteamine had no effect on the prolactin concentration of the irradiated rats. The results suggest that administration of WR-2721 or cysteamine prior to the irradiation has a potent preventive effect on theinitiation phase during mammary tumorigenesis.     

Thyroid status is a key modulator of tumor oxygenation: implication for radiation therapy

In normal tissues, thyroid hormones play a major role in the metabolic activity and oxygen consumption of cells. Because the rate of oxygen consumption is a key factor in the response of tumors to radiation, we hypothesized that thyroid hormones may affect the metabolic activity of tumor cells and hence modulate the response to cytotoxic treatments. We measured the influence of thyroid status on the tumor microenvironment in experimental tumors. Hypothyroidism and hyperthyroidism were generated in mice by chronic treatment with propyl thiouracil and l-thyroxine. Thyroid status significantly modified tumor pO(2) as measured with EPR oximetry. Mechanistically, this was the result of the profound changes in oxygen consumption rates. Thyroid status was associated with a significant change in tumor radiosensitivity since the regrowth delay was increased in hypothyroid mice compared to euthyroid mice, an effect that was abolished when temporarily clamped tumors were irradiated. This study provides unique insights into the impact of modulating tumor oxygen consumption and could have implications in the management of cancer patients with thyroid disorders.     

Increase in tumor oxygenation and radiosensitivity caused by pentoxifylline.

The effects of pentoxifylline (PTX), a drug commonly used for vascular disorders in humans, on the pO2 in SCK tumors of A/J mice and FSa-II tumors of C3Heb/FeJ mice as well as on the radioresponse of SCK tumors were investigated. When the host mice were injected intraperitoneally (ip) with 5 mg/kg PTX, the tumor pO2 increased slowly, peaked 20-50 min postinjection, and returned to its original level in 70-90 min. The magnitude of the increase in tumor pO2 varied markedly depending on the site and tumors. The magnitude of the changes in tumor pO2 after an ip injection of 25 or 50 mg/kg PTX was similar to that caused by 5 mg/kg PTX, but the pO2 tended to remain elevated longer with the higher dose of PTX. When the A/J mice bearing SCK tumors in the legs were injected ip with 50 mg/kg PTX and the tumors were X-irradiated 20 min later, the radiation-induced growth delay of the tumors was greater than that caused by X irradiation alone. The present study demonstrated that PTX is potentially useful for increasing the pO2 and the radioresponse of human tumors.     

Microbeam radiation therapy alters vascular architecture and tumor oxygenation and is enhanced by a galectin-1 targeted anti-angiogenic peptide

In this study, we sought to determine the therapeutic potential of variably sized (50 μm or 500 μm wide, 14 mm tall) parallel microbeam radiation therapy (MRT) alone and in combination with a novel anti-angiogenic peptide, anginex, in mouse mammary carcinomas (4T1)--a moderately hypoxic and radioresistant tumor with propensity to metastasize. The fraction of total tumor volume that was directly irradiated was approximately 25% in each case, but the distance between segments irradiated by the planar microbeams (width of valley dose region) varied by an order of magnitude from 150-1500 μm corresponding to 200 μm and 2000 μm center-to-center inter-microbeam distances, respectively. We found that MRT administered in 50 μm beams at 150 Gy was most effective in delaying tumor growth. Furthermore, tumor growth delay induced by 50 μm beams at 150 Gy was virtually indistinguishable from the 500 μm beams at 150 Gy. Fifty-micrometer beams at the lower peak dose of 75 Gy induced growth delay intermediate between 150 Gy and untreated tumors, while 500 μm beams at 75 Gy were unable to alter tumor growth compared to untreated tumors. However, the addition of anginex treatment increased the relative tumor growth delay after 500 μm beams at 75 Gy most substantially out of the conditions tested. Anginex treatment of animals whose tumors received the 50 μm beams at 150 Gy also led to an improvement in growth delay from that induced by the comparable MRT alone. Immunohistochemical staining for CD31 (endothelial cells) and αSMA (smooth muscle pericyte-associated blood vessels as a measure of vessel normalization) indicated that vessel density was significantly decreased in all irradiated groups and pericyte staining was significantly increased in the irradiated groups on day 14 after irradiation. The addition of anginex treatment further decreased the mean vascular density in all combination treatment groups and further increased the amount of pericyte staining in these tumors. Finally, evidence of tumor hypoxia was found to decrease in tumors analyzed at 1-14 days after MRT in the groups receiving 150 Gy peak dose, but not 75 Gy peak dose. Our results suggest that tumor vascular damage induced by MRT at these potentially clinically acceptable peak entrance doses may provoke vascular normalization and may be exploited to improve tumor control using agents targeting angiogenesis.     

Radiation induction of drug resistance in RIF-1 tumors and tumor cells

The RIF-1 tumor cell line contains a small number of cells (1-20 per 10(6) cells) that are resistant to various single antineoplastic drugs, including 5-fluorouracil (5FU), methotrexate (MTX), and adriamycin (ADR). For 5FU the frequency of drug resistance is lower for tumor-derived cells than for cells from cell culture; for MTX the reverse is true, and for ADR there is no difference. In vitro irradiation at 5 Gy significantly increased the frequency of drug-resistant cells for 5FU, MTX, and ADR. In vivo irradiation at 3 Gy significantly increased the frequency of drug-resistant cells for 5FU and MTX, but not for ADR. The absolute risk for in vitro induction of MTX, 5FU, and ADR resistance, and for in vivo induction of 5FU resistance, was 1-3 per 10(6) cells per Gy; but the absolute risk for in vivo induction of MTX resistance was 54 per 10(6) cells per Gy. The frequency of drug-resistant cells among individual untreated tumors was highly variable; among individual irradiated tumors the frequency of drug-resistant cells was significantly less variable. These studies provide supporting data for models of the development of tumor drug resistance, and imply that some of the drug resistance seen when chemotherapy follows radiotherapy may be due to radiation-induced drug resistance.     

A proof of principle experiment for microbeam radiation therapy at the Munich compact light source

Microbeam radiation therapy (MRT), a preclinical form of spatially fractionated radiotherapy, uses an array of microbeams of hard synchrotron X-ray radiation. Recently, compact synchrotron X-ray sources got more attention as they provide essential prerequisites for the translation of MRT into clinics while overcoming the limited access to synchrotron facilities. At the Munich compact light source (MuCLS), one of these novel compact X-ray facilities, a proof of principle experiment was conducted applying MRT to a xenograft tumor mouse model. First, subcutaneous tumors derived from the established squamous carcinoma cell line FaDu were irradiated at a conventional X-ray tube using broadbeam geometry to determine a suitable dose range for the tumor growth delay. For irradiations at the MuCLS, FaDu tumors were irradiated with broadbeam and microbeam irradiation at integral doses of either 3 Gy or 5 Gy and tumor growth delay was measured. Microbeams had a width of 50 µm and a center-to-center distance of 350 µm with peak doses of either 21 Gy or 35 Gy. A dose rate of up to 5 Gy/min was delivered to the tumor. Both doses and modalities delayed the tumor growth compared to a sham-irradiated tumor. The irradiated area and microbeam pattern were verified by staining of the DNA double-strand break marker γH2AX. This study demonstrates for the first time that MRT can be successfully performed in vivo at compact inverse Compton sources.     

Nitric oxide produced by inducible nitric oxide synthase is associated with mammary tumorigenesis in irradiated rats.

This study evaluated whether nitric oxide (NO) derived from nitric oxide synthase (NOS) induced by radiation is associated with tumorigenesis in the mammary glands. When rats were exposed to whole-body irradiation with gamma-rays (1.5 Gy) immediately after weaning and then treated with diethylstilbestrol, as an irradiated control, the tumor incidence (85%) was increased 7.6-fold in comparison with that (11.1%) of the non-irradiated control. The tumor incidence declined to 28.6% in the rats injected intraperitoneally with phenyl-N-tert-butylnitrone (PBN, 160 mg/kg), an inhibitor of inducible NOS (iNOS) expression and also a spin trapping agent, 30 min before irradiation. Also, the tumor incidence (25%) in rats orally administered with N-(3-(aminomethyl)-benzyl)-acetamide (1400W, 2.3+/-0.1 mg/day), a highly selective inhibitor of iNOS, dissolved in drinking water for 3 days after the irradiation was less than one-third of that in the irradiated control. On treatment with PBN or 1400W, no adenocarcinoma developed. Many of the mammary tumors that developed in the irradiated rats were positive for the estrogen receptor (ER). In contrast, ER was not detected in the tumors yielded from irradiated rats administered with PBN or 1400W. These results indicate that iNOS-derived NO may participate in the formation of estrogen-dependent mammary adenocarcinomas following radiation.     

Correlation of tumor oxygen dynamics with radiation response of the dunning prostate R3327-HI tumor

Our previous studies have shown that oxygen inhalation significantly reduces tumor hypoxia in the moderately well-differentiated HI subline of the Dunning prostate R3327 rat carcinoma. To test our hypothesis that modifying hypoxia could improve the radiosensitivity of these tumors, we performed experimental radiotherapy to compare the tumor response to ionizing radiation alone or in combination with oxygen inhalation. Tumor pO(2) measurements were performed on size-selected tumors several hours before radiotherapy using (19)F nuclear magnetic resonance echo planar imaging relaxometry (FREDOM) of the reporter molecule hexafluorobenzene. In common with our previous findings, the larger tumors (>3.5 cm(3)) exhibited greater hypoxia than the smaller tumors (<2 cm(3); P < 0.001), and oxygen inhalation reduced the hypoxic fraction (<10 Torr): In the larger tumors, hypoxic fraction dropped significantly from a mean baseline value of 80% to 17% (P < 0.001). The effect of oxygen administered 30 min before and during irradiation on tumor response to a single 30-Gy dose of photons was evaluated by growth delay. For the smaller tumors, no difference in growth delay was found when treatment was given with or without oxygen breathing. By contrast, breathing oxygen before and during irradiation significantly enhanced the growth delay in the larger tumors (additional 51 days). The differential behavior may be attributed to the low baseline hypoxic fraction (<10 Torr) in small tumors (20%) as a target for oxygen inhalation. There was a strong correlation between the estimated initial pO(2) value and the radiation-induced tumor growth delay (R > 0.8). Our histological studies showed a good match between the perfused vessels marked by Hoechst 33342 dye and the total vessels immunostained by anti-CD31 and indicated extensive perfusion in this tumor line. In summary, the present results suggest that the ability to detect modulation of tumor pO(2), in particular, the residual hypoxic fraction, with respect to an intervention, could have prognostic value for predicting the efficacy of radiotherapy.     

Suppression of thymic lymphoma induction by life-long low-dose-rate irradiation accompanied by immune activation in C57BL/6 mice

The induction of thymic lymphomas by whole-body X irradiation with four doses of 1.8 Gy (total dose: 7.2 Gy) in C57BL/6 mice was suppressed from a high frequency (90%) to 63% by preirradiation with 0.075 Gy X rays given 6 h before each 1.8-Gy irradiation. This level was further suppressed to 43% by continuous whole-body irradiation with 137Cs gamma rays at a low dose rate of 1.2 mGy/h for 450 days, starting 35 days before the challenging irradiation. Continuous irradiation at 1.2 mGy/h resulting in a total dose of 7.2 Gy over 258 days yielded no thymic lymphomas, indicating that this low-dose-rate radiation does not induce these tumors. Further continuous irradiation up to 450 days (total dose: 12.6 Gy) produced no tumors. Continuously irradiated mice showed no loss of hair and a greater body weight than unirradiated controls. Immune activities of the mice, as measured by the numbers of CD4+ T cells, CD40+ B cells, and antibody-producing cells in the spleen after immunization with sheep red blood cells, were significantly increased by continuous 1.2-mGy/h irradiation alone. These results indicate the presence of an adaptive response in tumor induction, the involvement of radiation-induced immune activation in tumor suppression, and a large dose and dose-rate effectiveness factor (DDREF) for tumor induction with extremely low-dose-rate radiation.     

Blood flow changes following 137Cs irradiation in a rat glioma model

Blood flow changes in response to 20 Gy 137Cs whole brain irradiation were measured with quantitative autoradiography of [14C]iodoantipyrine (IAP) in intracerebral grafts of the 36B-10 rat glioma, the brain around tumor (BAT), the contralateral corpus callosum, and the contralateral cerebral cortex. Irradiations were delivered on Day 14 post-transplantation, and measurements of flow (F) were performed with IAP on Day 15 or Day 16. Mean values of F were determined in individual tumors and in treatment groups. In 15- and 16-day-old unirradiated control tumors, the group mean F was 0.31 ml.g-1.min-1. In both 15- and 16-day-old tumor groups irradiated on Day 14 (Day 1 and 2 postirradiation tumors) the mean F for each day's group was 0.52 ml.g-1.min-1, 68% higher than the control (P less than 0.01). Flow in the BAT and the contralateral corpus callosum similarly was increased at these times (P less than 0.01). Flow in the contralateral cerebral cortex was 1.1, 1.5, and 1.3 ml.g-1.min-1 in the control, 1 day postirradiated, and 2 day postirradiated groups, respectively, but these increases were not significantly different from the control. These data indicate that flow increases in the intracerebral gliomas as well as in normal brain regions during the 2 days following 20 Gy irradiation. Changes such as these following radiotherapy may have important effects on the bioavailability of chemotherapeutic drugs.     

The oxygenation of murine tumor isografts and human tumor xenografts by nicotinamide.

The changes in pO2 caused by nicotinamide in the FSaII mouse tumor and three different xenografts of human tumors, HP-56, FaDu, and EO1, grown subcutaneously in the legs of mice were studied. The tumor pO2, as measured with microelectrodes, began to rise soon after the host mice were injected intraperitoneally with 500 mg/kg nicotinamide, and it increased continuously for 100-120 min. The rate and magnitude of the increase in tumor pO2 was dependent on the tumor line and also on the tumor size. In FSaII tumors, the increase in pO2 caused by nicotinamide was relatively small in the well-oxygenated small tumors (173 +/- 5 mm3) compared with that in the larger tumors (515 +/- 25 mm3). The blood perfusion in FSaII tumors as measured with the laser Doppler method was also increased by nicotinamide. The growth delay in FSaII tumors induced by X irradiation was enhanced significantly by nicotinamide. It was concluded that the enhancement of radiation damage in the experimental tumors in mice by nicotinamide, as observed in the present study and reported by others, is due to an increase in intratumor pO2, possibly as a result of an increase in blood perfusion.     

Irradiation-induced hypoxia in bones and soft tissues: an experimental study

Bone marrow and subcutaneous tissue pO2 and pCO2 were measured by means of implanted tissue tonometers in irradiated and nonirradiated rabbit hind limbs. The x-ray dose was 500, 1000, 1500, 2000, and 3000 rads. Tissue gas tensions were measured 1 day and 5 and 11 weeks after radiation. The pCO2 changes in both tissues were slight but not statistically significant. The subcutaneous tissue pO2 decreased during the acute phase of irradiation injury, and the effect of irradiation was dose-dependent. Later on, irradiation had no significant effects on the subcutaneous pO2, although light microscopy of the affected tissues showed fibrosis and blood vessel changes. The response of the subcutaneous pO2 to systemic hyperoxia also increased in the chronic phase of irradiation injury as a sign of improved microcirculation. The bone marrow showed a high radiosensitivity. Irradiation caused a rapid dose-dependent decrease of the marrow pO2, and the marrow pO2 decreased with time during the chronic phase of irradiation injury. The marrow pO2 responded slowly and marginally to an increment of arterial pO2 during breathing 100% oxygen as further evidence of impaired vascular pattern. The results showed that irradiation causes only a transient impairment of tissue perfusion in the skin. However, irradiation-damaged marrow was characterized by progressive tissue hypoxia.     

Tissue repair and repopulation in the tumor bed effect

These experiments were designed to study the kinetics and magnitude of cell repair and repopulation in tissues whose damage results in the tumor bed effect. The right hind thighs of mice were irradiated with single doses or two equal gamma-ray fractions. Interfraction intervals ranging from 30 min to 24 h (to measure the kinetics of repair from sublethal damage) and 6 and 12 weeks (to determine the extent of repopulation) were used. One day after the second radiation dose 5 X 10(5) FSA tumor cells were inoculated into the center of the irradiated field. Radiation dose-response curves were obtained by calculating the time required for tumors to reach 12 mm diameter. No recovery occurred within 6 h of the radiation delivery as measured by this assay. Some recovery, 3.2-4.6 Gy above a single radiation dose, occurred when the interval between two fractions was 24 h. With increasing interfraction intervals of 6 and 12 weeks further dose sparing occurred in the amount of 5.0-6.9 and 7.5-8.3 Gy, respectively. The data suggest that repopulation is the major contributor to the radiation dose-sparing recovery of stromal tissue and that some proliferative response may occur as early as 1 day after the first irradiation.     

SERUM LEVELS OF COLONY STIMULATING FACTOR(S) AND THE GROWTH OF BONE MARROW CELLS IN DIFFUSION CHAMBERS

Fluctuations in the body fluids of long-ranged humoral substance(s) capable of stimulating the growth of bone marrow granulocytic and macrophage-like cells in diffusion chamber cultures in vivo, was observed after whole body irradiation of mice. The fluctuation pattern was similar to that of the in vitro colony stimulating factor(s) of the sera of irradiated mice which indicates a relation between in vivo and in vitro active factor(s).     

Selection of genetically distinct,rapidly proliferating clones does not contribute to repopulation during fractionated irradiation in FaDu squamous cell carcinoma

Acceleration of clonogen repopulation during fractionated irradiation after about 3 weeks has been demonstrated previously in FaDu human squamous cell carcinoma in nude mice (Petersen et al., Int. J. Radiat. Oncol. Biol. Phys. 51, 483-493, 2001). Selection of genetically distinct, rapidly proliferating clones might contribute to this phenomenon. To address this question, three sublines (R1-R3) were established from FaDu tumors that recurred locally after fractionated irradiation. The tumors were retransplanted and irradiated under clamp hypoxia with single doses or with 18 x 3 Gy within 18 days or 36 days, followed by graded top-up doses. The results were compared with data obtained after the same treatment schedules in the parental tumor line. Histologies, tumor volume doubling times, and potential doubling times of FaDu sublines R1-R3 were not different from those of the parental line. The radiation dose required to control 50% of the tumors (TCD(50)) after single-dose irradiation of 37-38 Gy was the same for the FaDu sublines R1-R3 and the parental tumor. The top-up TCD(50) values for the FaDu sublines R1-R3 after 18 fractions within 36 days were 14-17 Gy higher than those after 18 fractions within 18 days, indicating significant repopulation. The magnitude of this effect was not significantly different between the sublines R1-R3 or between these sublines and the parental FaDu tumors. The results indicate that selection of genetically distinct, rapidly proliferating clones does not contribute to the acceleration of repopulation during fractionated irradiation in poorly differentiated FaDu tumors.