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The response of KHT sarcomas to one, two, five or ten daily fractions of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), with and without misonidazole (MISO), was evaluated using delay of tumour regrowth as the measure of response. When CCNU was given as 2 dose fractions separated by 24 h rather than as a single treatment, no extra dose was necessary to achieve a particular level of damage, suggesting a lack of damage repair. With increasing fraction number, however, an increasing total dose of drug was required to achieve a given effect, presumably to compensate for proliferation. Increasing drug doses also were readily tolerated (almost twice the LD50/7 for a single dose of CCNU resulted in no deaths when given in a 10 fraction treatment) indicating a large sparing of normal tissue toxicity when CCNU treatments were fractionated. The addition of MISO enhanced the tumour response to CCNU in all treatment schemes. When single doses of CCNU were combined with 0.5 mg g-1 MISO, an enhancement ratio (ER) of approximately 1.5 was observed. This ER was maintained for all fractionated treatment schedules including the 10 daily fraction protocol. In addition, no loss of sensitization with increasing fractionation was observed when a lower dose of 0.2 mg g-1 MISO was combined with each of 5 or 10 daily fractions of CCNU. Similar experiments were performed to test the combination of cyclophosphamide (Cy) and MISO (0.5 mg g-1) in the RIF-1 tumour, again chemopotentiation was maintained with increasing fractionation. These results of combined MISO and fractionated chemotherapy are in contrast to the rapid loss of sensitization observed when MISO is used as a radiation sensitizer and combined with small doses of X-rays, thus providing in vivo evidence of the mechanistic difference between the effects of MISO used as a radiation sensitizer or chemopotentiator. Peripheral white blood cell counts performed on mice receiving 5 daily fractions of CCNU +/- MISO displayed no significant enhancement of normal tissue toxicity by MISO. Thus combining MISO with repeated low dose treatments of a chemotherapeutic agent results in a therapeutic gain.

Intracerebral (i.c.) and subcutaneous (s.c.) 9L tumours were treated simultaneously with various doses of the nitrosoureas, BCNU or CCNU, and 2.5 mmol kg-1 of misonidazole (MISO). After 24 h, tumours were removed, dissociated into single cell suspensions and the cells plated for colony formation. In both i.c. and s.c. tumours, no cell kill was observed after exposure to MISO alone, and no additional cell kill was observed when MISO was combined with either nitrosourea. If s.c. 9L tumours were clamped 30 min after i.p. injection of 2.5 mmol kg-1 MISO, then 2 h later the clamps were removed and the nitrosourea injected, an increase in cell kill was observed. This increase in cell kill was statistically significant (P less than 0.01) for each dose of BCNU administered, but not statistically significant (P greater than 0.05) for the moderate dose of CCNU administered. Clamping did not alter the colony forming efficiency of cells from untreated 9L s.c. tumours or from those treated with each drug alone. These data demonstrate that hypoxic cells are required for misonidazole to potentiate the cell-killing effects of the nitrosoureas and that s.c. 9L tumours contain no such cells.

The effect of combinations of the conventional chemotherapeutic agent 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and nitroimidazole radiation sensitizers was evaluated in female C3H mice. Tumour response to single-agent or combination therapy was assessed in a tumour growth-delay assay. In the KHT sarcoma the simultaneous addition of misonidazole (MISO) was found to increase significantly the tumour growth delay resulting from CCNU treatment. The observed enhancement ratios (ER) increased with MISO dose, and ranged from 1.3 to 1.9 for sensitizer doses of 0.25-1.0 mg/g. The combination of CCNU and 1.0 or 0.5 mg/g MISO in the RIF-1 tumour or the MT-1 tumour produced ERs of approximately 2.0 and approximately 1.5 respectively. In the KHT sarcoma a series of other nitroimidazole sensitizers, including Ro-05-9963, SR-2555, SR-2508 and metronidazole (METRO), were also evaluated at equimolar doses (5 mmol/kg) in combination with a 20mg/kg dose of CCNU. Unlike MISO, these compounds in general failed to enhance the CCNU cytotoxicity in this tumour model. However, SR-2508 did enhance the response of the RIF-1 tumour to large single doses of CCNU, though not as much as MISO. Normal-tissue toxicity was determined using peripheral white blood cell (WBC) counts 3 days after treatment. CCNU doses of 10-50 mg/kg given either alone or in simultaneous combination with 0.5 or 1.0 mg/g MISO were studied. WBC toxicity increased with CCNU dose, but the addition of MISO at either dose did not significantly enhance this normal-tissue toxicity.

Studies were performed to determine whether the radiation sensitizer misonidazole (MISO) could enhance the tumor response of the KHT sarcoma to a treatment combining fractionated radiotherapy and the chemotherapeutic agent 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). A single dose of CCNU (20 mg/kg) was given 24 hr prior to the start of a multi-fraction radiation protocol in which 10 fractions were delivered once a day in 12 days overall treatment time. Daily radiation doses ranged from 1.0 to 4.0 Gy. MISO was administered at a dose of 1.0 mmol/kg, either once as a chemopotentiator simultaneously with CCNU, or repeatedly as a radiosensitizer 30-40 min prior to each radiation dose. Tumor response to treatment was assessed using tumor regrowth delay as the end point. The results indicate that, at 1.0 mmol/kg, MISO failed to radiosensitize the tumors in each of the fractionation schedules evaluated; that is, there was no difference between the regrowth delay curves obtained when CCNU treatment was followed by fractionated radiation, administered either alone or with MISO prior to each radiation dose fraction. However, when a single dose of 1.0 mmol/kg MISO was combined with CCNU 24 hr prior to the start of radiotherapy, regrowth delay was increased for all fractionated radiation schedules, particularly, at the larger dose fraction sizes. Comparison of the dose response curves suggests that MISO, used as a chemopotentiator, effectively reduced the proportion of radiobiologically hypoxic cells in the tumors prior to the start of the radiation therapy. These findings indicate that chemopotentiation can be used effectively in a combination with fractionated radiotherapy.

Patients with ovarian cancer often respond well to combination chemotherapy initially but the majority eventually relapse when, with further treatment, the initially successful regimen proves ineffectual. The cause of such failures frequently has been attributed to the development of drug resistance. Although the mechanisms of acquired resistance in situ are still poorly understood, studies in vitro have shown that cells selected for resistance to one drug often exhibit cross-resistance to other seemingly unrelated agents, suggesting a somewhat generalised mechanism of resistance. We have studied the role of glutathione (GSH) and drug transport in determining the sensitivity to adriamycin (ADR) of a panel of human ovarian cell lines established directly from biopsies of patients with diverse treatment histories. These cell lines exhibited inherent differences in sensitivity to ADR by a dose factor of up to 3; a difference that was considerably less than what has been reported when cells were selected for drug resistance in vitro. The differences in drug sensitivity reported here among the various cell lines appeared to be unrelated to drug transport, in terms of both influx and efflux. Moreover, although these cell lines have a wide range of GSH content, there was only a poor correlation between drug sensitivity and cellular GSH content per se. However, when exposed to a clinically relevant dose of ADR, the GSH content of cell lines that were 'sensitive' decreased, whereas that of cell lines that were 'resistant' increased. To take these time-dependent changes in GSH into consideration, the area under the GSH content versus time curve (AUC), with and without ADR treatment, was calculated for each cell line. When this latter factor was included in the analysis, greatly improved correlations were found between GSH kinetic parameters and responses to ADR. In particular, ADR resistance was found to be closely correlated with the positive changes in absolute GSH AUC following ADR treatment (r = 0.92; P less than 0.01). Using 35S-labelled cysteine and methionine as tracers, it was found that the essential difference between the 'resistant' and 'sensitive' lines was that the 'resistant' lines had higher steady-state rates of GSH synthesis than the 'sensitive' lines. These results demonstrate that changes in cellular GSH concentration during treatment may be an important indicator of tumour cell response to ADR.

The effectiveness of localized X-radiation on the transplantable KHT sarcoma was studied in nonanesthetized C3H mice possessing blood carboxyhemoglobin (HbCO) levels similar to those observed in heavy smokers. HbCO values of 10% were induced in tumor-bearing animals, either acutely just prior to irradiation or chronically during tumor growth and irradiation, by allowing the mice to breathe gas mixtures containing carbon monoxide (CO) in air. Tumors were irradiated either with single doses of 1500, 2000 or 2500 rad or with seven 500 rad fractions given at 24 hr intervals. Tumor cell survival was determined using an in vivo lung colony or an in vitro agar colony assay. The results with single doses of radiation indicate that under conditions of both acute and chronic exposure, the presence of 10% HbCO in the blood of the mice at the time of irradiation increases the survival of tumor cells in the hypoxic region of the survival curve by a factor of 2. During the fractionated irradiation, tumor cell survival in the presence of a 10% blood HbCO level (induced either acutely or chronically) was found to be significantly higher than that observed in air breathing mice. The results indicate that HbCO levels, such as are observed in heavy smokers, result in a larger fraction of hypoxic tumor cells. These findings suggest that heavy smoking prior to treatment may worsen the prognosis of patients undergoing radiotherapy.

The potentiation of chemotherapeutic agents by radiation sensitizers has been extensively studied for several years. There is little doubt that the effectiveness of certain anti-cancer drugs, primarily alkylating agents, can readily be enhanced both in vitro and in vivo through the addition of a sensitizer. While enhanced effects have been observed in certain critical normal tissues, in general most animal model studies have demonstrated a therapeutic gain at large sensitizer doses. This approach to combination therapies therefore appears promising. Yet many questions concerning the interaction between chemotherapeutic agents and radiosensitizers, particularly in the area of mechanisms of action, still remain. This overview attempts to focus on some of these questions. Four aspects of modification of chemotherapy by nitroimidazoles are reviewed and discussed. These address the importance in chemopotentiation of i) hypoxia, ii) alterations in DNA damage and/or repair, iii) depletion of intracellular sulfhydryls and iv) modification of drug pharmacokinetics. It is concluded that: i) even though chemopotentiation can occur at intermediate oxygen levels, hypoxia ultimately plays a pivotal role, ii) no single unifying mechanism for chemopotentiation exists; alterations in drug pharmacokinetics, cellular SH levels and DNA damage/repair all are involved, the relative importance of each factor is dependent on the particular drug-sensitizer combination, iii) it is important to continue the evaluation of chemopotentiation under conditions mimicking clinically achievable sensitizer pharmacokinetics and iv) further investigations into more effective utilization of chemopotentiation are warranted.

A previous study by the same authors has reported the effect of acute reductions in the arterial partial pressure of oxygen (PaO2) on tumour response to radiation. The results have been extended in the present paper to investigate tumour response to radiation in animals in which the PaO2 is chronically reduced. The purpose of these experiments was to simulate the condition of cancer patients undergoing radiotherapy in the presence of chronically low PaO2 values as might be expected in patients with chronic respiratory disease. Mice bearing transplantable KHT sarcomas were kept in a 12% O2 environment prior to (10--16 days), during and following the radiation treatment of their tumours. During the period of low PaO2 (about 50 mm Hg) exposure, the mice were found to increase their haemoglobin (Hg) levels by approximately 50%. Because of this increase, the response, determined using a growth delay assay of the tumours irradiated at reduced PaO2 was found to be the same as that observed for tumours in mice breathing air throughout the experiment. In mice with reduced PaO2 levels maintained at normal Hb concentrations by periodic bleeding, tumour response was found to be similar to that of mice with acute PaO2 reductions. These results indicate that chronic PaO2 reductions in the absence of Hb compensation may have a detrimental effect on the success of a radiation treatment.

In vitro potentiation of BCNU activity in rat brain tumour cells pretreated with misonidazole

The effect of localized radiation on the transplantable KHT sarcoma in unanesthetized C3H mice was investigated while the mice breathed either oxygen (O 2 ) or 5% CO 2 : 95% O 2 (carbogen) for times ranging from 0.5 to 120 min prior to irradiation. Tumors were irradiated with a single dose of 2000 rad at a dose rate of 1140 rad/min and tumor cell survival was determined using a lung colony assay. The results for O 2 breathing indicate that survival varies as a function of pre-irradiation breathing time (PIBT). Whereas 10 and 30 min give survival values three times lower than air survival, 15–20 min and very long times (1–2 hr) yield results which are similar to those obtained under air breathing conditions. In animals breathing carbogen there is a minimum in the survival level (factor of three lower than air survival) after 10 min of gas exposure followed by a slow rise in survival over the next 90 min at which time there is no sensitization by this gas mixture relative to air. During a course of seven 500 rad fractions given daily with carbogen, a 5 min PIBT was found to give significantly greater sensitization than a 0.5 min PIBT. These findings give a clear indication that the PIBT of O 2 and carbogen has a strong influence on the effectiveness of the radiation treatment and suggest the need to control this factor carefully in the clinic.

There recently has been considerable interest in combining conventional anti-tumor agents and nitroimidazole radiation sensitizers as a new approach in the treatment of tumors. This interest stems at least in part from the observations that oxygen-deficient or hypoxic tumor cells may be preferentially spared by certain chemotherapeutic agents because (i) these cells may be relatively inaccessible to the drug because they are located in poorly vascularized areas of the tumor, (ii), hypoxia per se may reduce the efficacy of the chemotherapeutic agent, or (iii) under hypoxic conditions a large proportion of cells may be in a noncycling state which protects the cells from cell cycle specific agents.33 On the other hand nitroimidazoles such as misonidazole (MISO) have been shown to possess, besides their radiation sensitizing ability, a direct cytotoxic effect against hypoxic cells.‘,’ ‘.25,29.44.47,50.54 Consequently, it was suggested33,47 that if hypoxia resulted in a chemo-protective effect, then the combination of two agents, one aimed against the oxic tumor cell population (chemotherapeutic agent), the other against the hypoxic tumor cells (nitroimidazole sensitizer), might enhance the treatment efficacy. That hypoxic conditions may modify the response to certain chemotherapeutic agents has been well-documented in cell culture systems’7.‘9.2’,33 and multicellular spheroids.7.4X.49.55 Less information is available for in vivo chemotherapy, although some reports of the occurrence of this phenomenon in mouse tumors treated with conventional anti-tumor agents such as BCNU* and nitrogen mustard have been described.‘*,” Initial demonstrations of enhanced cell killing resulting from combinations of chemotherapeutic agents and radiation sensitizers came from in vitro investigations. Previously it had been shown that hypoxic cells, exposed to radiation sensitizers such as MIS0 under conditions of prolonged incubation prior to irradiation, were more readily killed than would have been predicted solely on the basis of the radiation sensitization mechanism.‘0.62.h3 Subsequently it was shown that pre-treating cells under hypoxic conditions with MIS0 prior to the administration of anti-cancer agents such as bleomycin,33,‘4 adriamycin,4R melphalan, mustine and cisplatinum45 also markedly reduced cell survival. These “pre-incubation experiments” usually consisted of exposing cells under hypoxic conditions to fairly high doses of MIS0 (3-5 mM) for a few hours (spheroids were exposed for up to 24 hours), washing out the sensitizer, and then treating the cells under aerobic conditions either with various doses of the chemotherapeutic agent for a fixed time period or with a fixed drug dose for a variable time period. Compared to treatments with the chemotherapeutic agents alone, such sensitizer pre-treatments led to steeper slopes and/or smaller shoulders on the cell survival curves, and when evaluated by isobologram analysis to supra-additive cell kill in spheroids. Following these initial observations, chemo-potentiation in vitro has been reported by many investigators for various chemotherapeutic agents and radiation sensitizers (for review see Int. J. Radiat. Oncol. Biol. Phys. 8, No. 314, 1982). The first in vivo demonstrations of enhanced tumoricidal effects resulting from the combined treatments of radiosensitizers and conventional chemotherapy were reported in 1980 by Clement et ~1.~ and Rose et ~1.~~ Using in vivo to in vitro excision or in situ tumor regrowth assays, these authors showed that combining MIS0 with anti-tumor agents such as melphalan or cyclophosphamide (CYC) could result in enhancement ratios (ERs) of up to 2.5. Of special interest in these studies was the finding of a smaller enhancement of these agents by MIS0 in bone marrow stem cell toxicity (ER = 1.3-l .5) than in the tumor. Since these initial observations a variety ef combinations of chemotherapeutic agents and sensitizers have been tested in a number of different mouse tumor models (for review see Int. J. Rudiut. Oncol.

Eighteen adult colorectal cancer patients, previously untreated with systemic chemotherapy, were given CCNU and MISO. One patient had an excellent partial response of pulmonary metastases, but the overall response rate was only 6%. Gastrointestinal toxicity was modest, hematologic toxicity was similar to what would have been predicted for CCNU alone, and there was no neurotoxicity detected. This Phase II study demonstrates that these two agents can be administered safely, but have no advantage over CCNU alone.

Alterations in the oxygen (O 2 ) distribution in a tumor due to changes in the quantity of O 2 carried in the blood can affect the response of a tumor to radiation. For example, the blood hemoglobin (Hb) level has been shown to be an important prognostic and therapeutic factor in radiation therapy. Another factor factor affecting the delivery of O 2 to tissues in the Hb affinity for O 2 . Changes in Hb affinity for O 2 result in shifts of the Hb-O 2 dissociation curve which increase or decrease tissue oxygenation. The aim of the present studies was to determine whether reductions in Hb affinity prior to irradiation could improve the resultant tumor response. KHT sarcomas were irradiated in female C3H/HeJ mice, possessing either normal or reduced Hb affinities for O 2 at the time of treatment. Changes in Hb affinity for O 2 were induced by keeping tumor-bearing mice in a 12% O 2 environment for various periods of time. Erythrocyte 2,3 diphosphoglycerate (2,3 DPG) was measured as an indicator of Hb affinity for O 2 . After 36 hr of low O 2 exposure, 2,3 DPG levels increased 20–30%. This change in 2,3 DPG reflected a proportional decrease in Hb affinity for O 2 . Following the exposure of 12% O 2 , the animals were removed from the low 02 chamber and their tumors locally irradiated while the mice breathed air. After irradiation, tumor cell survival was determined using an in vivo to in vitro excision assay. The results indicate that the fraction of hypoxic cells in tumors of mice whose Hb affinity had been reduced prior to irradiation was ∼3%. By comparison, the hypoxic fraction in tumors irradiated in mice with normal Hb affinities was ∼15%. Thus, reductions in Hb affinitty prior to irradiation can yield significant radiation sensitization in tumors. These findngs form the basis for future investigations of the use of pharmacologic methods for the in vivo alteration of the HbO 2 dissociation curve to improve tumor oxygenation.

The effect on intramuscularly growing KHT sarcomas of combinations of the chemotherapeutic agent cyclophosphamide (CYC) and the radiosensitizer misonidazole (MISO) was determined. Tumor response was evaluated using an in vivo to in vitro clonogenic cell survival assay or an in situ tumor growth delay assay. MISO, at doses ranging from 0.25 to 1.0 mg/g, was found to be effective at enhancing the tumor response to CYC when given for times of 0–4 hr before the chemotherapeutic agent. Clonogenic cell survival studies in which a 1.0 mg/g dose of MISO was administered simultaneously with CYC demonstrated that the addition of the sensitizer increased the level of tumor cell kill assessed 24 hr after treatment by a factor of ∼4–8 for CYC doses ranging from 25–150 mg/kg. Similarly, in the in situ experiments MISO enhanced the regrowth delay resulting from CYC treatments with doses up to 150 mg/kg by ∼2–4 days. Consequently, the addition of MISO to CYC was not found to be strictly dose modifying, but rather resulted in enhancement ratios ranging from 1.3 to 1.5 depending on the dose of the chemotherapeutic agent used. Normal tissue toxicity to combinations of CYC and MISO was assessed 24 hr after treatment using a spleen colony assay. When MISO (1.0 mg/g) was administered simultaneously with a range of CYC doses, a dose modifying factor of ∼1.2 for bone marrow stem cell toxicity was observed. On the basis of these observations, the addition of MISO to CYC treatment may lead to a small therapeutic gain.

We have investigated in detail the effects of buthionine sulfoximine (BSO), a selective glutathione (GSH) depleting agent, on the GSH contents of a number of normal tissues and three experimental tumours in mice. Significant variations in the rate and degree of GSH depletion and recovery were observed among the normal tissues. Following a dose of 2.5 mmol kg-1 BSO, GSH nadirs were reached by approximately 5 h for the liver and kidney, 8 h for the lung and bone marrow, 12 h for red blood cells (RBCs) and by 24 h for the heart. The degree of depletion was greatest for the kidney (80%), liver (74%) and bone marrow (83%), intermediate for the heart (54%) and lung (40%), and least for RBCs (13%). Recovery of GSH content was fastest for the liver followed in descending order by the kidney, the lung, the bone marrow, RBCs and the heart. In contrast, the rate and extent of GSH depletion and recovery showed considerably less variation among the 3 murine tumours. In the tumours GSH nadirs were reached by 10-12 h. The extent of depletion was about 55-65%. Recovery of GSH levels in the tumours required 48 h or more, a longer period than required by the liver, kidney and lung but shorter than that needed for the bone marrow, heart and RBCs. Attempts to preferentially deplete tumour GSH by exploiting the differences in recovery rates between normal tissues and tumours were only partially successful. Multiple BSO dosing at 16 h intervals allowed the liver to recover between doses, but the recovery in the kidney, lung and bone marrow was only partial and no recovery was seen in the heart. Finally, dose-depletion relationship investigations showed that, with the exception of the lungs, GSH depletion could be achieved in tumours with doses of BSO lower than those required for normal tissues.

Cells from 9L/Ro, KHT, and EMT6/Ro solid tumors were separated into relatively homogeneous sized populations by centrifugal elutriation using a modified long collection procedure. This modification substantially improved the volume distributions of cells isolated from these solid tumors. More than 90% of the host cells were removed before the tumor cells were separated according to their sedimentation velocities into subpopulations at different stages of the cell cycle. Analysis of the DNA content of these subpopulations by flow cytometry indicated that the degree of synchrony after elutriation could be as high as ⩾92% for G1 cells, 70–75% for S cells, and ⩾70% for G2 cells for some of these tumors. Similar results were obtained by autoradiography. Nevertheless, the recovery in these fractions was often low, varying from 0.3 to 10% of the total number of cells elutriated. The colony-forming efficiency of cells from different phases of the cell cycle remained constant and was always higher than that of unseparated tumor cells, because there were few host cells in the separated populations.

The human colon adenocarcinoma cell line WiDr was xenografted and the tumor characterized. When athymic mice (NCR-nu) were inoculated with 10(6) cells, tumors appeared after 7-14 days with a 93-100% take rate and grew with an initial volume-doubling time of around 6 days. For optimizing the tumor disaggregation method, a comparison was made of two dissociation procedures and of different dissociation times. An enzyme cocktail (collagenase, DNase, pronase) resulted in total viable cell yields of 1-3 X 10(7) cells/g tumor tissue. Cell yield decreased with increasing tumor weight. Disaggregation with trypsin gave lower cell yields; and so, although the plating efficiencies (PEs) were higher, the enzyme cocktail was chosen for tumor disaggregation. On the basis of morphologic identification, cell suspensions prepared from WiDr tumors, by use of the enzyme cocktail for 2 hours, contained 49% malignant cells as well as a significant fraction of nonneoplastic cells. The major nonneoplastic host cell component was macrophage (33%); lymphocytes (13%) and granulocytes (5%) also were present. Host cells could be separated from neoplastic cells by centrifugal elutriation. By mixing various proportions of host and tumor cells, it was subsequently shown that the presence of host cells did not influence the malignant cell PE unless the cell suspensions contained greater than 90% host cells. Single-cell suspensions prepared from WiDr tumors, with use of the enzyme cocktail for 2 hours, were irradiated and then plated for survival (D0 = 1.5 Gy; n = 5) (D0, the 37% dose slope). A comparison was made of the sensitivity to radiation, after the different dissociation methods. The radiation sensitivities after 1.5-hour trypsinization and 2- and 6-hour enzyme cocktail administrations were similar, but after 0.5 hour of trypsin, the cells were more sensitive to radiation.

Previous studies using the KHT sarcoma have shown that misonidazole (MISO) enhances the cytotoxicity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) by as much as a factor of 2.0, In the present study flow cytometry was used to monitor the changing DNA distributions of cells dissociated from solid tumors at successive times following treatment with CCNU, applied either alone or in combination with 0.5 mg/g MISO. The proportion of cells in late S and the G 2 M phases of the cell cycle increased gradually after CCNU treatment. MISO did not significantly change this block in cell progression, which persisted for at least 48 hr after treatment in all cases. CCNU shows marked carbamoylating activity, which has been associated with inhibition of RNA processing and with the degree of chemopotentiation achieved with MISO. Consequently, to evaluate whether MISO chemopotentiation was influencing the RNA distributions in tumors, RNA histograms were generated using acridine orange to differentially stain cellular DNA and RNA. By 24 hr after treatment. CCNU clearly altered the distribution of RNA, but no significant differences could be detected between results obtained from drug and drug plus sensitizer treated groups. These studies demonstrate the effect of CCNU on cell cycle progression in vivo . The addition of MISO did not result in further perturbation of the total tumor population, suggesting that cell cycle redistribution does not play a major role in chemopotentiation by MISO.

The effectiveness of localized X-irradiation on the transplantable KHT sarcoma was investigated while the host mice breathed gas mixtures ranging from 5% O2 : 95% N2 to 100% O2. Subcutaneous tumours in the flank of C3H mice anaesthetized with Nembutal (sodium pentobarbitol), Valium (diazepam), or urethane, were irradiated with doses of 1,500 or 2,500 rads and the delay in days for the tumours to grow from 4 to 10 mm in diameter was determined. Measurements of the arterial partial pressure of oxygen (PaO2) were made under conditions identical to the irradiation experiments. For PaO2 values less than 100 mm Hg (animals breathing air), growth delay decreased linearly, while for PaO2 values greater than 100 mm Hg, growth delay increased and reached a plateau at a PaO2 between 200 and 300 mm Hg, (animals breathing congruent to 50% O2). This relationship holds for the two doses and the three anaesthetics studied, and indicates the importance of investigating the Pa02 levels of patients undergoing radiotherapy treatment.

Publisher Summary This chapter discusses the separation of malignant cells from host cells using centrifugal elutriation. Several cell separation procedures have been developed for the purification of in situ host cells from tumor cells. The chapter reviews the application of centrifugal elutriation as a procedure for the isolation of different cell subpopulations from tumor tissue in situ. In the process of cell separation by centrifugal elutriation, cells suspended in the separation chamber of an elutriator rotor are subjected to two opposite forces: the centrifugal force generated from the rotation of the rotor in an outward direction and the fluid force through the chamber in an inward or centripetal direction. The special shape of the separation chamber makes it possible to generate a gradient of flow rate, decreasing toward the center of rotation. Various practical aspects of the separation involved in the method of centrifugal elutriation can influence the homogeneity of cell subpopulations. The chapter also discusses those factors that are directly relevant to the separation of in situ tumor cells. Some of these factors include cell volume distributions between the host and malignant cells, effects of tumor dissociation on the quality and quantity of single-cell suspensions, and the modification of collection methods to improve the homogeneity.

The effect of including a nitroimidazole in a treatment regimen combining two alkylating chemotherapeutic agents was evaluated in a mouse tumor model. KHT sarcoma-bearing female C3H/HeJ mice were treated with a single dose of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) either alone or in combination with the radiosensitizer misonidazole (MISO) prior to a range of cyclophosphamide (CY) doses. CCNU (5 mg/kg) preceded CY treatment by 3 hr. MISO (1 mmol/kg) was given 1 hr after CCNU (2 hr prior to CY). Tumor response was assessed using either an in vitro to in vivo clonogenic cell survival assay 22-24 hr after treatment or an in situ delay of tumor regrowth assay. The results demonstrated that the inclusion of MISO at a dose of 1.0 mmol/kg increased tumor cell kill resulting from the combination of CCNU-CY by a factor of 1.4-1.5 compared to that seen in the absence of the nitroimidazole. Normal tissue toxicity resulting from the CCNU-CY or CCNU-MISO-CY combinations was determined by measuring bone marrow stem cell (CFU-S or CFU-GM) toxicity. Compared to CCNU-CY alone, the addition of MISO did not enhance this normal tissue toxicity. These findings indicate that the inclusion of MISO in a combination chemotherapy protocol may yield a significant therapeutic benefit.

Abnormal vascularization often associated with tumor growth may result in the presence of significant numbers of radiation resistant hypoxic cells in some solid tumors, thereby limiting the effectiveness of radiotherapy in cancer treatment. Sufficiently low levels of oxygen have been well-documented as causing radiation resistance of mammalian cells in culture and in tumors in animals. There is also a significant amount of data available suggesting the presence of hypoxic resistant cells in human tumors. Hypoxic cells in tumors are not abnormally oxygenated because of the large diffusion distances from blood vessels and intervening consumption of oxygen by other tumor cells. One area of major research emphasis in the past ten years has been the search for compounds which sensitize hypoxic cells to ionizing radiation but are not readily consumed by the tumor cells and with pharmacological properties which result in sensitizing concentrations in regions of hypoxic cells in poorly vascularized tumor tissue.

In order to determine the role of recovery of tumor cell survival in the enhanced tumor responses resulting from chemotherapeutic agent-sensitizer combinations, cell survival in KHT sarcomas as a function of time after treatment with the alkylating agents 1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), cyclophosphamide (CYC), phenylalanine mustard (L-PAM) and 2-[3-(2-chloroethyl)-3-nitrosoureidol-D-glucopyranose (chlorozotozin; CLZ) either alone or in combination with the radiosensitizer misonidazole (MISO) was evaluated. Tumor-bearing mice were treated with single doses of CCNU, BCNU, CYC, L-PAM and CLZ, respectively. The doses, chosen to obtain approximately the same level of cell kill with each treatment, were administered either alone or in simultaneous combination with a 1.0 mg/g dose of MISO. Tumors were excised at 1, 2, 4, 8, 16 and 24 hr after drug injection. Clonogenic cell survival was assessed at these times using an in vivo to in vitro excision assay. The results showed that following treatment with each of the chemotherapeutic agents, tumor cell survival reached a minimum at ∼2–8 hr. Subsequently cell survival remained relatively constant for CYC, but increased with time by a factor of ∼10–100 for L-PAM, CCNU, BCNU and CLZ. The addition of MISO potentiated the cell killing effects of CYC, L-PAM, CCNU and BCNU, but not of CLZ. Also, in animals treated with certain of these chemotherapeutic agents plus the sensitizer, cell survival recovery was impaired. This effect was particularly marked when the nitrosourea CCNU was used in combination with MISO (i.e. 24 hr after treatment with CCNU plus MISO, tumor cell survival was a factor of ∼50 lower than observed in tumors of mice given only CCNU). These findings indicate that the enhancement of cell killing in the KHT sarcoma observed 24 hr after treatment with chemotherapeutic agent-MISO combinations may be at least in part the result of altered recovery in tumor cell survival.

The immediate release of surfactant into lung alveoli following irradiation has been studied as a potential indicator for the later development of radiation pneumonitis. Utilizing single dose radiation exposure to the whole thorax in male LAF1/J mice, steep dose response curves for lavaged alveolar surfactant were identified at 7 and 28 days after exposure. Seven days after irradiation there was no elevation with doses up to and including 12 Gy; above this dose a detectable increase occurred. At 28 days the surfactant recovered by lavage was elevated compared to the levels seen at day 7 for all doses; doses greater than 12 Gy produced surfactant values significantly greater than those found in mice treated with 12 Gy or less. The radiation pulmonary lethality dose response curve assessed four months later indicated an LD50 value of approximately 13 Gy. The early biochemical effect and the later radiation pneumonitis lethalities therefore closely coincided. The evidence strongly indicates that alveolar surfactant release uncovered hours to days after radiation exposure may be an early biochemical marker that predicts for subsequent pneumonitis radiation injury.

We have used monochlorobimane as a quantitative marker by which cells of naturally high or low GSH contents were purified by fluorescence-activated cell sorting (FACS). The cell line chosen for this purpose, MLS, was a human ovarian tumor cell line established from a patient who had received extensive chemotherapy and showed evidence of 'multidrug' resistance. Cells of a specified volume were sorted into subpopulations containing the 1% most dim (low GSH) and 1% most bright (high GSH) cells. With an increasing number of sortings, cell subpopulations emerged with progressively lower (dim) and higher (bright) GSH content as compared to the parent population. After 4 sortings, GSH contents were 10.6 +/- 0.8, 5.1 +/- 0.4, and 7.2 +/- 0.7 X 10(-18) moles/micron3 for MLS/bright, MLS/dim and MLS/parent respectively. The high and low GSH phenotypes were of limited stability reverting to the parent phenotype by the sixth week following the last FACS. Cells with high GSH content were more resistant to adriamycin than cells with low GSH content, for example, at 1 log cell kill MLS/bright was 1.6 fold more resistant than MLS/dim. An ADR resistant variant of the MLS line, designated MLS/ADRR/2, established by twice treating MLS cells with 1 microgram/ml ADR for 2 hr, also showed increased GSH content (1.3-fold) and ADR resistance as compared with the parent line. These results illustrate the possible importance of tumor cell GSH status in determining the response to chemotherapy of a heterogenous population of tumor cells with diverse GSH contents.

The problem of applying experimental tumor studies to clinical cancer therapy is a complex one. The radiotherapy literature contains many examples of premature efforts to apply laboratory observations to the clinic, and many examples of failures to adequately consider animal tumor observations in the design of clinical studies. This review covers three areas: tumor hypoxia, where clinical trials based on animal tumor data have been conducted with radiosensitizers, hyperbaric oxygen, and systemic oxygen carriers; dose fractionation, where current trials of hyperfractionation are based in part on animal tumor studies; and chemo-radiotherapy, where clinical trials are only beginning to exploit concepts developed in animal tumor systems. The use of animal tumor systems extends past the screening of new agents. Animal tumor models can be used in biological, physiological, pharmacological studies to elucidate the biological factors influencing the efficacy of therapeutic agents. Tumor studies can be combined with studies of normal tissues to predict the toxicities to be anticipated in clinical trials, and to assess the potential for therapeutic gain. Animal studies can also provide data whic are useful in designing optimal clinical trials of new agents and maximizing the potential for successful clinical application of new approaches. In general, it is not possible to apply specific laboratory data directly to man. To translate, rather than transpose, information from the laboratory to the clinic, the model studies must be directed at evaluating principles, rather than merely quantifying results. Only through studies of mechanisms, by designing experiments to test or refute a hypothesis, will it be possible to apply model studies to man.

Adriamycin (ADR) to a total dose of 11 mg/kg LD 10 60 ) was administered intraperitoneally to female Balb/cKa mice either as a single dose or in multiple dose fractions. The concentration of ADR equivalents as a function of time after injection was determined using a fluorometric assay in the heart, lung, liver, kidney and intramuscularly growing EMT-6/Ro tumor. The results for single doses indicate both the maximum attainable tissue drug level and retention kinetics to be tissue specific. At 1–3 hr post-injection, measured values were highest in the liver and lowest in the tumor. Adriamycin equivalents had a longer half-life ( t 1 2 ) in the tumor (67 hr) than in the other tissues examined: lung, (45 hr), liver (29 hr), kidney (27 hr), and heart (20 hr). Studies also were performed to determine whether the ADR pharmacokinetics are altered during a multiple dose regimen. Mice were given either a single dose of 5.5 mg/kg, 2 daily doses of 2.75 mg/kg, or 5 daily doses of 1.1 mg/kg. The results showed: 1) a build-up in tissue ADR equivalents with a maximum value after each fraction dependent solely on the level of drug remaining from the previous dose and 2) that in all tissues studied, the pharmacokinetics of the subsequent dose of ADR are unaffected by both the size and number of previous dose increments.

The KHT sarcoma is a model system in which metastases can be studied in multiple organs without prior clonal selection. The present series of experiments were designed to evaluate and compare the extent of potentiation of chemotherapeutic agent activity by radiosensitizers when KHT sarcoma cells were grown in different anatomical sites. In these studies the effect of combining misonidazole (MISO) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) on KHT lung nodules and ovarian metastases was determined. Ovarian metastases were a consequence of the direct spread of tumor cells growing as lung nodules. Once established, KHT cells in the lungs and ovaries grew with a similar doubling time (1-2 days). Response of tumors at either site to chemotherapy in the presence or absence of a sensitizer was assessed using an in vivo to in vitro excision assay. MISO (1.0 mmol/kg) was administered simultaneously with a range of CCNU doses and survival of clonogenic tumor cells was measured 24 h after treatment. The results demonstrate that the addition of MISO to CCNU treatment potentiated the action of this chemotherapeutic agent at both tumor sites although greater cell kill enhancement occurred in the ovarian metastases. In the lung nodules, when combined with CCNU, a 1.0 mmol/kg dose of MISO was found to yield a dose modifying factor (DMF) of approximately 1.3. The same combination resulted in a DMF of approximately 1.8 in the ovarian metastases. This difference in DMF was not a result of an intrinsic difference in sensitivity to CCNU since cells grown at either site gave rise to the same dose response curve. Rather the difference in dose modification by MISO appears to be a consequence of the larger fraction of radiobiologically hypoxic cells in the ovarian tumors (approximately 50 per cent) than in the lung nodules (approximately 5 per cent) at the time of treatment. These findings suggest the use of drug-sensitizer combinations to treat disseminated disease and provide further evidence for the requirement of hypoxia in chemopotentiation by radiosensitizers.

The effect of combining the radiosensitizer misonidazole (MISO) and the chemotherapeutic agent 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) on mouse lung tumors of different sizes and hypoxic fractions was tested. KHT sarcoma lung colonies were treated at volumes of ∼0.7 and 2.0 mm 3 . At these sizes the lung colonies contained ∼5 and 15% hypoxic tumor cells respectively. For the smaller lung colonies response to CCNU or the CCNU-MISO combination was evaluated by determining the percent of mice tumor-free 100 days after treatment. This assay could not be used when 2.0 mm 3 lung colony bearing mice were treated, since at this size only a few animals could be cured with either the single or combined modality therapy. Consequently, tumor response in these experiments was assessed using an increase in lifespan assay or a tumor growth delay assay. When 0.7 mm 3 KHT lung colonies were treated with CCNU plus 1.0 mg/g MISO, the TCD50100 (dose to cure 50% of the mice of their tumors in 100 days) was reduced by a factor of 1.5–1.8 compared to mice receiving only CCNU. However, because the single agent therapy was less toxic than the combination, increasing doses of CCNU ultimately resulted in the same level of cure as could be achieved with the CCNU-MISO combination. In the 2.0 mm 3 lung colonies studies, however, when compared at the maximally tolerated doses, the addition of 0.5 or 1.0 mg/g MISO to CCNU led to a percent increase in median lifespan, which was a factor of 1.4–1.5 greater than could be achieved with CCNU alone. This increase in lifespan was shown, using growth delay studies, to be a direct consequence of an enhanced tumoricidal response to the combination therapy. These findings indicate that in this lung tumor model an improved therapeutic result may be achieved through the addition of MISO to treatment with CCNU.

The response of murine EMT6/Ro tumors and KHT sarcomas to adriamycin (ADR) given either as a single dose, 2 equal doses or multiple dose increments (all to the same total dose) was determined using a tumor growth delay assay. The total ADR uptake in the tumors was similar for the various drug schedules. An ld 10 60 dose of ADR (lethal to 10% of the mice in 60 days) led to a growth delay equivalent to one doubling time in these established tumor lines as well as a first generation transplanted mammary tumor. Two doses, each equal to half the ld 10 60 dose, separated by 1–7 days or 1–2 days, were as effective against the EMT6/Ro tumor and KHT sarcoma respectively as a single ld 10 60 dose. These findings suggest a lack of repair of ADR damage in these tumors. However, such a total dose ( ld 10 60 ), given as multiple daily increments, resulted in a growth delay less than that following a single ld 1 60 exposure likely due to cell repopulation.

RSU 1069, the lead compound in a series of nitroimidazoles containing an alkylating aziridine function, has been shown to be a potent radiosensitizer and chemopotentiator both in vitro and in vivo . However, this agent also demonstrates significant in situ toxicity. Recently it has been shown that less toxic analogues of RSU 1069 can be produced by the introduction of alkyl substituents to moderate the reactivity of the aziridine function. The present investigations were undertaken to evaluate the in vivo cytotoxicity and chemosensitizing efficacy of two such analogues, RSU 1164 and RSU 1165. All experiments were performed with KHT sarcomas grown intra-muscularly. In the cytotoxicity studies, a range of sensitizer doses was utilized whereas in the chemopotentiation investigations a fixed sensitizer exposure was combined simultaneously with a range of doses of the nitrosourea CCNU. In both studies, tumor cell survival was determined 22–24 hr after treatment using a soft agar clonogenic assay. Normal tissue toxicity in the chemopotentiation studies was assessed by bone marrow CFU-S assay. Both analogues were found to be significantly :less cytotoxic to KHT sarcoma cells than RSU 1169 (a factor of 4–6 in dose at 50% cell survival). Combining a 1.0 to 2.0 mmol/kg dose of RSU 1164 or RSU 1165 with a range of doses of CCNU increased tumor cell killing by a factor of 1.5–1.6 but did not enhance bone marrow stem cell toxicity. The addition of either sensitizer to CCNU treatment therefore led to a significant therapeutic benefit.

Studies were performed to evaluate the radiation response across the cell cycle of KHT sarcoma cells grown as solid tumours. Centrifugal elutriation, a relatively non-perturbing method for synchronising cells, was used to obtain homogeneous populations of cells with respect to their positions in the cell cycle from suspensions prepared directly from KHT tumours. More than 90% of the non-neoplastic cells were removed and the tumour cells separated into fractions containing 90-95% Gj cells, 70-75% S cells and 70-75% G2M cells. Since survival following in situ irradiation is influenced by the presence and proportion of oxygen-deficient or hypoxic tumour cells, in order to study the radiation response across the cell cycle, KHT sarcoma cells were irradiated under conditions of uniform oxygenation. This was achieved by irradiating the tumour cells either in situ in dead animals or, in vitro, after tumour dissociation and cell separation, while fully oxic or anoxic. Under all these conditions, the most radioresistant portion of the cell cycle was found to be late G! and early S. G 2M cells were always most radiosensitive. Complete radiation dose-response curves under oxic or anoxic conditions were also obtained for cells in all phases of the cell cycle. From these the oxygen enhancement ratio (OER) was calculated to be 2.4, 2.6 and 2.5 for cells in the Gj, S and G2M cell cycle phases respectively.

Studies were performed to determine the location, with respect to cell cycle phase, of the radiobiologically hypoxic cells in KHT sarcomas. Cells dispersed from solid KHT tumours were separated into subpopulations at different stages of the cell cycle by centrifugal elutriation. Flow cytometric analysis of the DNA content of these subpopulations indicated that the degree of synchrony that could be achieved was greater than or equal to 95% for G1 cells, 70-75% for S cells and 70-75% for G2M cells. The approach to locate and characterize hypoxic cells was based on the premise that due to their lack of oxygen such cells would preferentially survive radiation treatment. Consequently KHT sarcomas were irradiated in situ either in dead animals or in animals breathing air. Following treatment, the tumours were dissociated, the cells elutriated into the various phases of the cell cycle and clonogenic cell survival was determined. Complete dose-response curves were determined for cells in the G1, S and G2M cell cycle phases. The various cell cycle subpopulations obtained from tumours irradiated while mice breathed air, all demonstrated survival curves with a break and final slope which paralleled that of the corresponding anoxic cell survival curve. From these curves the proportion of hypoxic tumour cells in the G1 phase was calculated to be 10.1 +/- 1.7%. Because the elutriated S and G2M enriched cell fractions were to some extent contaminated by cells from other phases of the cell cycle, the percentage of hypoxic S and G2M tumour cells was estimated to range from 0-7% and 0-5% respectively. However, since G1 cells comprised the majority of all the neoplastic cells in these tumours, the data suggest that hypoxic cells in KHT sarcomas are found primarily in this cell cycle stage.

Highly electron affinic compounds such as the nitroimidazole misonidazole (MISO) have been shown both in vitro and in vivo to be effective potentiators of certain conventional chemotherapeutic agents. Mechanistically, the observation that nitroheterocyclics reduce intra-cellular thiols by enhancing the oxidation of glutathione (GSH), has suggested that thiol depletion by MISO may be a key factor in this enhancement. The present investigations were undertaken to determine whether the use of buthionine sulfoximine (BSO) to affect GSH metabolism may lead to more effective potentiation of chemotherapeutic agents by sensitizers. KHT/iv cells were treated in exponential phase under hypoxic conditions with variable doses of the activated form of cyclophosphamide (4-hydroxy-cyclo-phosphamide, 4OH-CY) administered concomitantly with or without MISO (2.5 mM) for an exposure time of 4 hr. Inclusion of the sensitizer in the treatment protocol resulted in a dose modifying factor of approximately 2.4. Exposing cells to 1.0 mM BSO for 2 hr prior to treatment reduced intracellular GSH levels to 70-80% of control and increased the efficacy of 40H-CY approximately 1.2-fold. If BSO was administered prior to the 4OH-CY + MISO combination, severe tumor cell toxicity resulted. For example, when combined with 4OH-CY, similar cell kill could be achieved with 5 to 6-fold lower MISO doses in the presence of BSO as in the absence of BSO. Ultrastructural evaluations revealed that in the three agent combination, membrane damage, as reflected by the formation of surface blebs, may play a key role in the mechanism of the observed enhanced cytotoxicity.

The response of intramuscularly growing KHT sarcomas to the chemotherapeutic agent (1-(2-cloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) alone or simultaneously with the chemical radio-sensitizer misonidazole (MISO) was assessed using either a tumour growth-delay assay or an in vivo-in vitro tumour-excision assay. Median tumour growth delay following the combination of 20 mg/kg CCNU and either 0.5 or 1.0 mg/g MISO was 19.5 and 21.5 days, compared to 10 days for this CCNU dose alone. A similar degree of enhanced tumour response by MISO (factor of approximately 2 in tumour growth delay) was seen in RIF-1 tumours treated with 20 mg/kg CCNU plus 1.0 mg/g MISO. Clonogenic cell-survival studies with KHT sarcomas demonstrated that MISO at doses of 0.25, 0.5 or 1.0 mg/g given simultaneously with a range of CCNU doses produced dose-modifying factors (DMFs) of 1.9, 2.1 and 2.4 respectively. Normal tissue toxicity assessed by an LD50/7 assay led to DMFs of 1.2 and 1.4 for CCNU doses combined with 0.5 and 1.0 mg/g MISO. Thus in this animal tumour model the combination of CCNU and MISO appears to lead to a potential gain by a factor of approximately 1.7.

Metastatic renal cell carcinoma (RCC) associates with overproduction of vascular endothelial growth factor (VEGF) due to the mutation/inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. Herein we demonstrate that implantation of human RCC tumor cells into athymic nude mice promotes the appearance of VEGF receptor 1 (VEGFR1)/CD11b double-positive myeloid cells in peripheral blood. Avastin-mediated VEGF neutralization was capable of significantly reducing the numbers of circulating VEGFR1+ myeloid cells. Conversely, up-regulation of VEGFR1 by myeloid cells could also be achieved in vitro by coculturing bone marrow cells with RCC-conditioned medium or by short-term exposure of naive myeloid cells to oxidative stress. Treatment of myeloid cells with H2O2, lipid peroxidation product 4-hydroxy-2(E)-nonenal, or an inhibitor of thioredoxin reductase all resulted in increased expression of VEGFR1. Furthermore, after exposure to oxidative stress, myeloid cells acquire immunosuppressive features and become capable of inhibiting T cell proliferation. Data suggest that tumor-induced oxidative stress may promote both VEGFR1 up-regulation and immunosuppressive function in bone marrow-derived myeloid cells. Analysis of tumor tissue and peripheral blood from patients with metastatic RCC revealed that VEGFR1+ cells can be also found in cancer patients. Restoration of immunocompetence in metastatic RCC patients by pharmacological elimination of VEGFR1+ cells may have a significant impact on the therapeutic efficacy of cancer vaccines or other immune-based therapies.

Purpose: To determine whether electrode measurements of tumor oxygenation, made in a variety of murine tumor models, correlate with estimates of radiobiological hypoxia in the same tumor systems. Methods and Materials: The tumor models used were a C3H mammary carcinoma grown in the feet of CDF1 mice; the SCCVII, KHT and RIF-1 tumors grown in the feet or flanks of C3H/Km mice; and the CaNT and SaF tumors grown on the backs of CBA mice. All treatments were performed when tumors were about 200 mm 3 in size. Radiobiological hypoxic fractions were determined using either a paired survival curve assay, with survival measured 0–24 h after irradiation, or using a clamped tumor control assay, with percent local tumor control estimated 90 days after treatment. Measurements of tumor oxygen partial pressure (pO2) distributions were performed using Eppendorf oxygen electrodes. Results: The hypoxic fractions determined from the radiation response data were about 1% in RIF-1 and SCCVII, 12% in C3H and KHT, 28% in CaNT and up to 38% in SaF tumors. When this data was compared with the tumor oxygenation measurements it was found that as hypoxic fraction increased the mean, median, and the percentage of pO 2 values ≤ 5 mmHg showed a trend towards poorer oxygenation status. However, none of these pO 2 changes were significantly correlated with hypoxia. Moreover, the pO 2 values ≤ 2.5 mmHg indicated an improvement in oxygen status with increasing hypoxic fraction. Conclusion: Electrode measurements of tumor oxygenation alone may, therefore, not be a good indicator of tumor hypoxia across different tumor cell lines.

Combinations of single and multiple doses of adriamycin and radiation were studied in EMT-6/Ro tumor-bearing BALB/cKa mice. The response to these chemotherapy-radiotherapy treatments was determined using a tumor growth delay assay. The single-dose interaction between adriamycin and radiation was assessed using an isoeffect analysis and was found to be additive for both a 2- and 120-hr interval between agents when changes in tumor size were accounted for. When a single dose, two equal doses, or multiple daily doses of adriamycin (all to the same total dose) were combined with a course of ten 200-rad fractions given in 12 days, the resultant tumor growth delays also were in keeping with an additive response between the two treatment modalities. However, in combination with fractionated radiotherapy, a large single dose of adriamycin was more effective than concurrent small daily adriamycin exposures.

The lungs of 2 to 3-month-old female C3H/HeJ and 6-month-old male LAF1 mice were locally irradiated with graded doses of radiation ranging from 900 to 1800 rad. The patterns of animal details for times up to 60 weeks after irradiation were recorded. Over this period mice in all the irradiated groups continued to die. However, the onset of animal deaths was dose dependent such that lethality occurred earlier after the higher radiation doses. Whereas mice receiving large radiation doses started to die by 75-100 days post-treatment, few or no animal deaths occurred in those groups receiving the lowest doses until 200-300 days after irradiation. At 180 and 420 days postirradiation animal lethality dose-response curves were constructed; these were found to be similar in shape (sigmoid), but at the later time the calculated LD/sub 50/ values were approx.300-400 rad lower than those determined using the 180-day post-treatment end point. Histological examination of the irradiated lungs indicated that the animal deaths occurring by 180 days were associated with radiation pneumonitis while the late deaths were associated with pulmonary fibrosis. The addition of the radiation sensitizer misonidazole prior to lung irradiation did not significantly enhance pulmonary toxicity at either end point.

The response of KHT sarcoma cells growing as microscopic lung colonies to radiation treatments given with or without the hypoxic cell sensitizer misonidazole was evaluated using an in vivo to in vitro clonogenic cell survival assay. For comparison the survival curve for well-oxygenated KHT sarcoma cells was obtained by excising the colony-containing lungs from their hosts, dispersing the lung colonies to a single-cell suspension, and irradiating the cells in vitro. By increasing the time between excision and irradiation from 2 to 24 hr it was possible to demonstrate for these lung colony-derived cells an intercellular contact effect denoted by a reduction in the shoulder of the cell survival curve over this period. In vivo radiation treatments were given at two different lung colony volumes, 0.7 and 1.8 mm/sup 3/. The addition of misonidazole prior to irradiation sensitized the hypoxic cell compartment at both colony sizes and reduced the final slope in each case to the same degree (factor of approx. 1.8). At a cell survival level of 10/sup -3/ this resulted in an enhancement ratio of approx. 1.2 to approx. 1.5. By comparison this dose of misonidazole did not enhance the radiation-induced pulmonary toxicity determined using a LD/sub 50/180/ assay.

The arterial pH and partial pressures of oxygen (PaO/sub 2/) and carbon dioxide (PaCO/sub 2/) were evaluated in LAF1 mice 15 and 38 weeks after localized irradiation of the animals' thoraxes. Graded radiation doses of 900 to 1200 rad were administered. These doses resulted in 0 to 100% lethality by 26 weeks (180 days) after irradiation. At 15 weeks after treatment mice receiving radiation doses which would subsequently result in lethality (by 180 days) exhibited significant reductions in their PaO/sub 2/ and elevations in their PaCO/sub 2/ values, respectively. However, there was no clear dose-response relationship between blood gas values and radiation dose, which may reflect the animals' ability to compensate for their poor blood gas exchange by an increased breathing frequency. At 38 weeks after irradiation the blood gas values were abnormal in mice from groups which had normal blood gas values at Week 15 (and no fatalities by Week 26) but in which animal deaths had occurred between Weeks 26 and 38. These data therfore indicated (i) that abnormal blood gas values occurred in the mice prior to fatalities resulting from the acute radiation pneumonitis syndrome and (ii) that mice surviving the initial radiation pneumonitis phase could still more » succumb to progressive pulmonary toxicity which was reflected by the increasing levels of animal lethality and altered blood gas tensions at the later times. « less