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The c-Met/HGF pathway is implicated in cancer progression and dissemination. Many inhibitors have been developed to target this pathway. Unfortunately, most trials have failed to demonstrate efficacy. However, clinical trials have not adequately tested the concept of c-Met pathway inhibition due to the lack of appropriate patient selection criteria.

The tumor microenvironment is increasingly recognized as a major factor influencing the success of therapeutic treatments and has become a key focus for cancer research. The progressive growth of a tumor results in an inability of normal tissue blood vessels to oxygenate and provide sufficient nutritional support to tumor cells. As a consequence the expanding neoplastic cell population initiates its own vascular network which is both structurally and functionally abnormal. This aberrant vasculature impacts all aspects of the tumor microenvironment including the cells, extracellular matrix, and extracellular molecules which together are essential for the initiation, progression and spread of tumor cells. The physical conditions that arise are imposing and manifold, and include elevated interstitial pressure, localized extracellular acidity, and regions of oxygen and nutrient deprivation. No less important are the functional consequences experienced by the tumor cells residing in such environments: adaptation to hypoxia, cell quiescence, modulation of transporters and critical signaling molecules, immune escape, and enhanced metastatic potential. Together these factors lead to therapeutic barriers that create a significant hindrance to the control of cancers by conventional anticancer therapies. However, the aberrant nature of the tumor microenvironments also offers unique therapeutic opportunities. Particularly interventions that seek to improve tumor physiology and alleviate tumor hypoxia will selectively impair the neoplastic cell populations residing in these environments. Ultimately, by combining such therapeutic strategies with conventional anticancer treatments it may be possible to bring cancer growth, invasion, and metastasis to a halt.

Purpose Intratumoral hypoxia is known to be associated with radioresistance and metastasis. The present study examined the effect of acute and chronic hypoxia on the metastatic potential of prostate cancer PC-3, DU145, and LNCaP cells. Methods and Materials Cell proliferation and clonogenicity were tested by MTT assay and colony formation assay, respectively. “Wound-healing” and Matrigel-based chamber assays were used to monitor cell motility and invasion. Hypoxia-inducible factor 1 alpha (HIF-1α) expression was tested by Western blot, and HIF-1-target gene expression was detected by real-time polymerase chain reaction. Secretion of matrix metalloproteinases (MMPs) was determined by gelatin zymography. Results When PC-3 cells were exposed to 1% oxygen (hypoxia) for various periods of time, chronic hypoxia (≥24 h) decreased cell proliferation and induced cell death. In contrast, prostate cancer cells exposed to acute hypoxia (≤6 h) displayed increased motility, clonogenic survival, and invasive capacity. At the molecular level, both hypoxia and anoxia transiently stabilized HIF-1α. Exposure to hypoxia also induced the early expression of MMP-2, an invasiveness-related gene. Treatment with the HIF-1 inhibitor YC-1 attenuated the acute hypoxia-induced migration, invasion, and MMP-2 activity. Conclusions The length of oxygen deprivation strongly affected the functional behavior of all three prostate cancer cell lines. Acute hypoxia in particular was found to promote a more aggressive metastatic phenotype.

The vasculature of solid tumors is fundamentally different from that of normal vasculature and offers a unique target for anti-cancer therapy. Direct vascular-targeting with Tumor-Vascular Disrupting Agents (Tumor-VDAs) is distinctly different from anti-angiogenic strategies, and offers a complementary approach to standard therapies. Tumor-VDAs therefore have significant potential when combined with chemotherapy, radiotherapy, and angiogenesis-inhibiting agents. Preclinical studies with the different Tumor-VDA classes have demonstrated key tumor-selective anti-vascular and anti-tumor effects.

Purpose: The goal of this study was to examine the therapeutic potential of the vascular endothelial growth factor (VEGF) signaling inhibitor cediranib in a human model of renal cell carcinoma (Caki-1). Methods and Materials: The effects of cediranib treatment on in vitro endothelial cell function (proliferation, migration, and tube formation), as well as in vivo angiogenesis and tumor growth, were determined. Results: In vitro, cediranib significantly impaired the proliferation and migration of endothelial cells and their ability to form tubes, but had no effect on the proliferation of Caki-1 tumor cells. In vivo, cediranib significantly reduced Caki-1 tumor cell-induced angiogenesis, reduced tumor perfusion, and inhibited the growth of Caki-1 tumor xenografts. Conclusions: The present results are consistent with the notion that inhibition of VEGF signaling leads to an indirect (i.e., antiangiogenic) antitumor effect, rather than a direct effect on tumor cells. These results further suggest that inhibition of VEGF signaling with cediranib may impair the growth of renal cell carcinoma.

The purpose of this study was to investigate two non-invasive methods for determining the treatment efficacy of the vascular disrupting agent (VDA) CA4P: gadolinium enhanced dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for perfusion analysis and enzyme-linked immunosorbent assay (ELISA) of blood samples. Candidate proteins were identified by multi-analyte profile analysis of plasma from KHT sarcoma-bearing C3H/HeJ mice after CA4P administration. Candidate proteins were further analysed by ELISA of plasma from treated C3H/HeJ, BALBc and C57BL6 mice. Changes in selected proteins, tumour perfusion and tumour necrotic fraction after CA4P treatment were then compared in individual animals. The cytokines KC and MCP-1 were observed to increase after CA4P treatment in all tested models. No correlation was found between KC or MCP-1 levels and tumour necrosis. However, tumour perfusion correlated (r=0.89, p0.00001) with CA4P treatment efficacy as measured by necrotic fraction, suggesting that DCE-MRI may have utility in a clinical setting.

Purpose: To examine the pathophysiologic impact of treatment with combretastatin A4 phosphate (CA4P) in regions of tumors that ultimately either necrose or survive treatment with this agent. Methods and Materials: Proliferation, perfusion, vessel density, and expression of vascular endothelial growth factor (VEGF) were analyzed in the KHT tumor model after treatment with CA4P. Analyses were conducted in the whole tumor and the tumor periphery. Results: Perfusion in the tumor periphery decreased 4 h after treatment, but returned to baseline 20 h later. Whole-tumor perfusion also decreased 4 h after treatment, but did not return to baseline. Vessel density decreased in the tumor as a whole, but not in the tumor periphery. No significant effect on the expression of VEGF was observed, but a decrease in proliferation in the whole tumor and the periphery was noted. Conclusions: The present study shows that those areas of a tumor that survive treatment with CA4P are affected by CA4P exposure, though only transiently. The decrease in perfusion could negatively affect therapies utilizing the combination of CA4P and conventional anticancer agents by decreasing drug delivery and tissue oxygenation. These findings suggest that the timing of CA4P treatments when used in conjunction with conventional anticancer therapies should be considered carefully.

The present study evaluated the treatment efficacy of the vascular disrupting agents CA4P and OXi4503 in an orthotopically transplanted human renal cell carcinoma xenograft model (Caki-1). Experiments used vascular casting, vessel density assessments as well as tumour necrosis measurements to evaluate the efficacy of these agents. After treatment with either agent, assessment of the vascular casts showed an almost total eradication of tumour blood vessels. Histological evidence further supported this observation, showing extensive central tumour necrosis with only a small viable rim of tumour cells remaining at the periphery. These results suggest that vascular disrupting agents CA4P and OXi4503 may have utility in the treatment of renal cell carcinoma, an encouraging result given that current conventional therapies have been currently largely unsuccessful in managing this disease.

Background and purpose The present studies were undertaken to determine whether 19 F MRS could be used to quantify the binding of the pentafluorinated derivative of etanidazole (EF5) in hypoxic cells of solid tumors. Materials and methods A 4.7T imaging magnet was used for the in situ and in vitro evaluation of EF5 signals. In order to develop a better understanding of these NMR measurements the characteristics of parent, reduced unbound, and reduced bound EF5 signals were examined in vitro using a 12T spectrometer. Results In situ data acquired using a 4.7T imaging magnet, showed retention of EF5 signals in KHT sarcomas that was absent in muscles for 6h after EF5 injection. In vitro studies showed no difference in the NMR detectable signal of parent and reduced unbound EF5. T 2 values determined using parent EF5 samples revealed a T 2 time of 675ms. In contrast, EF5 bound to KHT tumor cells gave rise to signals of low intensity, broad line widths, and T 2 relaxation times of less than 30ms. When the same samples were analyzed using the 4.7T imaging magnet, the CF 3 and CF 2 fluorine peaks were readily identifiable in the parent EF5 sample but no fluorine signal could be detected from EF5 bound to KHT tumor cells. Conclusion The inability to resolve bound EF5 metabolites even at high field strengths (12T), coupled with the short T 2 relaxation times of the bound EF5, and the limits of detection of the in situ applied imaging magnet (4.7T), meant that hypoxic cells could not be quantified in tumors using the 19 F MRS technique. In situ 19 F MRS measurements of EF5 signals (parent/reduced unbound) may reflect conditions of tumor physiology and thus indicate the extent of tumor hypoxia but they are not capable of resolving the cellular oxygenation status of the tumor cells.

This articles discusses therapeutic strategies that selectively target and disrupt established tumor vasculature.

To evaluate whether sustained expression of mouse endostatin by adeno-associated virus (AAV)-mediated gene transfer can enhance the treatment efficacy of ionizing radiation.Mouse endostatin was cloned into recombinant AAV (rAAV) under the control of CMV beta-actin promoter. Recombinant mouse endostatin expressed via AAV gene transfer was tested for biological activity in endothelial cells. The impact of elevated serum levels of endostatin on tumor-induced angiogenesis was evaluated using an in vivo angiogenesis assay. The anti-tumor efficacy of combining rAAV-mediated endostatin delivery with radiation was evaluated in a human colorectal tumor model (HT29).Recombinant mouse endostatin expressed through an AAV vector (rAAV-mEndo) inhibited endothelial cell proliferation (by 40-45%) and migration (by 22-33%). Intramuscular injection of rAAV-mEndo (1x10(9) i.u.) led to a sustained serum endostatin level of approximately 500 ng/ml. Compared to control animals this endostatin level was sufficient to inhibit tumor cell-induced vessel formation (37 vs. 28.5, P0.05) and delay the growth of HT29 xenografts (time from 200 to 1,000 mm(3), 21 vs. 34.5 days, P0.05). When combined with ionizing radiation, elevated serum endostatin levels significantly enhanced the time for tumors to grow from 200 to 1,000 mm(3) (radiation, 34 days; endostatin plus radiation, 50 days, P0.05).The delivery of endostatin via rAAV vectors may provide an effective means of enhancing the anti-tumor efficacy of radiation therapy.

Purpose: To discuss the role of postoperative radiation therapy (RT) for patients with squamous cell carcinoma of the head and neck. Results: Patients with adverse pathologic features have a high likelihood of local-regional recurrence and a decreased probability of survival after surgery alone. Postoperative RT reduces the risk of local-regional failure and probably improves survival. Patients who are at high risk for recurrence after surgery benefit from more aggressive dose-fractionation schedules that may include altered fractionation to decrease the overall time from surgery to the completion of RT. Adjuvant chemotherapy also appears to improve the probability of cure in high risk patients. Conclusion: Patients who have a high likelihood of local-regional recurrence after surgery have improved disease control and survival after postoperative RT. (Am J Otolaryngol 2003;24:41-50. Copyright 2003, Elsevier Science (USA). All rights reserved.)

Purpose : The chemosensitizing potential of the benzotriazine- N -oxide tirapazamine was determined in rodent mammary tumor cells grown as solid tumors. Materials and methods : C3H/HeJ mice bearing i.m. transplanted 16C mammary carcinomas were treated with varying doses of either cisplatin alone or cisplatin in combination with a 0.27 mmol/kg dose of tirapazamine. Tumor response to single agent or combination therapy was assessed using an in situ tumor growth delay assay. Normal tissue toxicity resulting from the tirapazamine, cisplatin, or tirapazamine plus cisplatin was determined by measuring bone marrow stem cell (CFU-GM) toxicities and blood urea nitrogen (BUN) levels. Results : Tirapazamine itself had no measurable effect on the growth of this tumor. However, when administered from 3 h before to simultaneously with a single dose of cisplatin, the resultant tumor growth delay was significantly increased as compared to that seen with cisplatin alone. The administration of tirapazamine 3 h prior to a range of doses of cisplatin was found to result in a dose modifying factor (DMF) of ~1.7 in tumor response compared to cisplatin alone. Tirapazamine did demonstrate some hematologic toxicity on its own but it did not potentiate the toxicity of cisplatin when the two agents were administered in combination. BUN analysis showed that tirapazamine had little effect on BUN levels but did suppress the BUN values of mice treated with the combination of tirapazamine and 15 mg/kg cisplatin as compared to cisplatin alone. Conclusions : The present findings suggest that the addition of tirapazamine to cisplatin therapy may lead to a therapeutic benefit.

Purpose: To investigate whether electrode measurements of tumor oxygenation obtained under a range of different treatment conditions designed to alter the degree of tumor hypoxia could be correlated with estimates of radiobiological hypoxia measured under the same conditions. Methods and Materials: Experiments were performed in restrained, nonanesthetized, female C3H/He mice, which had ∼0.5 g KHT sarcomas growing intramuscularly in the hind limbs. The treatments used to modify tumor oxygenation status included breathing gas mixtures of varying oxygen content, altering tumor blood flow, and shifting the hemoglobin oxygen dissociation curve. Radiobiological hypoxic fraction was estimated using the paired survival curve assay, while electrode measurements of tumor oxygenation were obtained with an Eppendorf histograph. Results: With the selected manipulations it was possible to vary the radiobiological hypoxic fraction in the tumors from ∼1 to ∼100% of the total viable cell population. Furthermore, these changes in radiation response were directly reflected in the changes in tumor oxygenation measurements made with the Eppendorf histograph. Conclusion: These findings suggest that in the KHT tumor model the Eppendorf electrode measurements could predict the response of the tumors to radiation as determined by the proportion of hypoxic cells.

PURPOSE The aim of these studies was to determine whether chemotherapy-resistant tumor cell sublines derived from a single starting cell population with identical treatment protocols, have the same mechanism of resistance. METHODS AND MATERIALS Twelve cyclophosphamide-resistant sublines were derived from KHT-iv murine sarcoma cells by repeated exposures to 2, 4, or 8 microg/ml doses of 4-hydroperoxycyclophosphamide (4-OOHCP). To investigate possible mechanisms of resistance, glutathione (GSH) levels, glutathione S-transferase (GST) activity, and aldehyde dehydrogenase (ALDH) activity were determined. In addition, studies with the GSH depletor buthionine sulfoximine (BSO) and the ALDH inhibitor diethylamino-benzaldehyde (DEAB) were undertaken. RESULTS Resistant factors to 4-OOHCP, assessed at 10% clonogenic cell survival, ranged from 1.5-7.0 for the various cell lines. Crossresistance to melphalan and adriamycin also were commonly observed. Increased GSH levels, GST activity and ALDH activity were detected in the sublines but not all exhibited the same pattern of biochemical alterations. The response to GSH and ALDH inhibitors also varied among the sublines; the resistance being reversible in some cell lines but not others. CONCLUSION The present results indicate that when resistant sublines are derived simultaneously from the same starting cell population, the observed mechanisms of resistance may not be the same in each of the variants. These findings support the hypothesis that preexisting cellular heterogeneity may affect mechanisms of acquired resistance.

Background and purpose : Nicotinamide is a radiation sensitizer currently undergoing clinical testing. This was an experimental study to determine the importance of drug dose and time interval between drug administration and irradiation for radiosensitization. Materials and methods : Nicotinamide (50–500 mg/kg) was injected intraperitoneally into CDF1 or C3H mice and drug plasma pharmacokinetics were determined by HPLC. Radiosensitization was measured in tumours and normal tissues after local irradiation. The tumours were a C3H mammary carcinoma, the KHT sarcoma and the SCCVII carcinoma. Tumour response was assessed using either growth delay (C3H) or clonogenic survival (KHT/SCCVII). Normal tissue toxicities evaluated included early responding skin (development of moist desquamation of the foot) and late responding bladder (reservoir function estimated by cystometry) and lung (ventilation rate measured by plethysmography). Results : All nicotinamide peak plasma concentrations were seen within 30 min after injection. Irradiating tumours at peak times resulted in enhancement ratios (ERs) of 1.27 (C3H), 1.75 (KHT) and 1.45 (SCCVII) with high nicotinamide doses and 1.27 (C3H), 1.28 (KHT) and 1.36 (SCCVII) after giving clinically relevant doses (100–200 mg/kg). Lower ERs were observed when the time interval between drug injection and irradiation was increased beyond the peak time. Irradiating normal tissues at peak times after injecting 100–200 mg/kg nicotinamide gave ERs of 1.20 (skin), 0.90 (bladder) and 1.02 (lung). Conclusions : Clinically achievable doses of nicotinamide will enhance tumour radiation damage while having minimal effects in normal tissues, but for the best tumour effect radiation should be given at the time of peak plasma drug concentrations.

Refractory disease is the greatest challenge in treating patients with acute myeloid leukemia (AML). Blood vessels may serve as sanctuary sites for AML. When AML cells were co-cultured with bone marrow endothelial cells (BMECs), a greater proportion of leukemia cells were in G0/G1. This led us to a strategy of targeting BMECs with tubulin-binding combretastatins, causing BMECs to lose their flat phenotype, degrade their cytoskeleton, cease growth, and impair migration despite unchanged BMEC viability and metabolism. Combretastatins also caused downregulation of BMEC adhesion molecules known to tether AML cells, including vascular cell adhesion molecule (VCAM)-1 and vascular endothelial (VE)-cadherin. When AML-BMEC co-cultures were treated with combretastatins, a significantly greater proportion of AML cells dislodged from BMECs and entered the G2/M cell cycle, suggesting enhanced susceptibility to cell cycle agents. Indeed, the combination of combretastatins and cytotoxic chemotherapy enhanced additive AML cell death. In vivo mice xenograft studies confirmed this finding by revealing complete AML regression after treatment with combretastatins and cytotoxic chemotherapy. Beyond highlighting the pathologic role of BMECs in the leukemia microenvironment as a protective reservoir of disease, these results support a new strategy for using vascular-targeting combretastatins in combination with cytotoxic chemotherapy to treat AML.

The cytotoxicity and chemosensitizing potential of the nitrohetercyclic agent RB 6145 and its R enantiomer PD 144872 were determined in rodent tumor cells grown in tissue culture or as solid tumors. Using a clonogenic cell survival assay the degree of selective cytotoxicity of these bioreductive drugs was first determined in KHT/iv cells. Cells treated under hypoxic conditions were observed to be ~50–80-fold more susceptible to the action of RB 6145 or PD 144872 than were cells exposed under air. To assess the in vitro chemosensitizing potential of RB 6145 and PD 144872, doses of these agents which led to survival values between 0.5 and 1.0 under hypoxic conditions were administered, and were then combined concomitantly with variable doses of the nitrosourea CCNU. Exposure to the nitrosourea was for 1 h. The results showed that inclusion of either sensitizer enhanced the cell killing of the chemotherapeutic agent 2.4-2.6-fold. Subsequent experiments evaluated the therapeutic benefit of combining these bioreductive agents with CCNU in KHT sarcoma-bearing C3H/HeJ mice. When given at times ranging from 90 min before to 60 min after CCNU exposure, these bioreductive drugs increased the tumoricidal activity of the chemotherapeutic agent. Complete dose response curves combining RB 6145 and PD 144872 and a range of CCNU doses also were evaluated. The sensitizers (0.75 mmol/kg) were administered 30 min prior to the chemotherapeutic agent and survival of clonogenic tumor cells 22–24 h after treatment was used to assay tumor response. Normal tissue toxicity under similar treatment conditions was assessed using a bone marrow stem cell (CFU-GM) assay. When combined with CCNU, the bioreductive agents increased tumor cell kill by a factor of ~1.7. Bone marrow toxicity was not, however, enhanced by the addition of either agent to the CCNU treatment. These data suggest that further consideration should be given to the clinical application of these bioreductive agents.

Preclinical investigations have demonstrated that both diffusion- and perfusion-limited hypoxic cells may exist in tumors. One approach to target such hypoxic cell subpopulations is through the combined application of nicotinamide (NIC) administration and carbogen (5% CO 2 :95% O 2 ) breathing. Because carbogen pre-irradiation breathing time (PIBT) can markedly influence the radiosensitizing effectiveness of this gas mixture, in the present experiments the effect of localized radiation on the transplantable KHT sarcoma was investigated in mice receiving NIC while breathing carbogen for various periods of time. When mice were given carbogen prior to radiation therapy, there was a minimum in tumor cell survival for PIBTs of 2–30 min. Longer PIBTs led to a loss of the radiosensitizing effect. NIC, administered as a 1000-mg/kg dose, effectively enhanced radiation cell killing in this tumor if given 45 min to 2 h prior to radiotherapy. In experiments in which either agent was combined on its own under optimum conditions (carbogen, 10 min PIBT; or NIC, 1000 mg/kg 2 h prior to irradiation), with a range of single doses of radiation, the results showed an enhancement ratio of ∼1.9 as determined from the ratio of the slopes of the cell survival curves obtained in the absence or presence of the radiation sensitizer. This sensitizing effect could not be increased further when NIC and carbogen breathing were combined under optimum conditions.

I t is now well accepted that the cycle phase and proliferation state of a cell may significantly affect response to radiation and cytotoxic drugs. The experimental basis for this belief comes primarily from in vitro studies using synchronized cells and/or exponential or plateau-phase cell cultures. Indeed, using a variety of synchronization techniques, extensive studies have been carried out to determine the cycle phase dependence of the killing action of radiation and a wide variety of anticancer drugs. E,2 Most of these investigations have described the age response function of a particular cell line treated with a given agent. Typically in such studies, cell subsets, synchronized at the various phases of the cell cycle, are treated with a fixed dose of the cytotoxic agent and cell survival as a function of cell cycle phase is determined (Fig 1). For radiation, most, although not all, cell lines have shown greatest radiation sensitivity in the M and late G2 phases and maximum resistance in the S phase. 35 In contrast, cell age response patterns for chemotherapeutic agents vary considerably, although most do show differential efficacy in some parts of the cell cycle. 1,2,6 To evaluate the impact of proliferation state on anticancer therapies, many investigators have compared the treatment efficacy of the test agent in log or plateau phase cell cultures. These growth phases are chosen to mimic in vitro the nutrient deprivation that can occur in tumors and cause the formation of subpopulations with distinct proliferation states. 7 The cell populations include a cycling or proliferative subpopulation (P cells), a noncycling or quiescent subpopulation (Q cells), and a nonproliferating sub

The cytotoxicity and chemosensitizing potential of four dual function nitrofurans was determined in human HT-29 multi-cell spheroids and rodent KHT sarcoma solid tumors. Spheroids were treated with a range of doses of the bioreductive drugs for a period of up to 48 h and the extent of cell kill was assessed at various times after treatment. Cytotoxicity was determined using a clonogenic cell-survival assay. The results demonstrated that two of the nitrofurans were even more toxic to spheroid cells than was the potent bioreductive nitroimidazole aziridine RSU 1069. The dose of the nitrofuran which, after a 24-h exposure, led to a survival value between 0.5 and 1.0, then was chosen for subsequent studies aimed at assessing the ability of these agents to potentiate the efficacy of the nitrosourea CCNU. Exposure to this chemotherapeutic agent was for a period of 1 h. The results indicated that all four dual function nitrofurans enhanced the cell killing of the conventional chemotherapeutic agent by factors ranging from 1.1 to 1.7. Subsequent studies evaluated the therapeutic benefit of combining these bioreductive agents and CCNU in KHT sarcoma-bearing C3H/HeJ mice. The nitrofurans were administered i.p. 0.5 h prior to the chemotherapy and tumor response was assessed by measuring the survival of clonogenic KHT cells 22–24 h after treatment. Normal tissue toxicity was determined using a bone marrow stem cell (CFU-GM) assay. Combining these bioreductive agents with CCNU increased the tumor cell kill by factors of 1.2 to 1.7. Bone marrow toxicity increased with CCNU dose, but the addition of the nitrofurans failed to enhance this endpoint of normal tissue toxicity.

The efficacy and selective hypoxic cell cytotoxicity of four dual function nitrofurans and two nitroimidazole-aziridines was determined in human (A549, HT-29) and rodent (KHT/iv) tumor cells. All bioreductive compounds were found to be less effective at killing human than mouse tumor cells (approximately 2-6-fold). This reduced cytotoxicity in the human tumor cells occurred irrespective of the state of oxygenation. In addition, the degree of selective toxicity toward hypoxic cells or the cytotoxicity factor (CF), defined as the ratio of the surviving fraction in air to that under hypoxic conditions, was (a) greater for the nitroimidazole-aziridines than for the nitrofurans and (b) less in the human than the rodent tumor cell lines investigated. For example, CF values in A549 or HT-29 cells typically were 2-4-fold lower than those determined in KHT/iv cells. This reduction in the CF in the human cells resulted from a greater loss in the hypoxic toxicity than in the aerobic toxicity when compared with the rodent cells.

Acquired drug resistance is a major obstacle in the successful treatment of cancer by chemotherapy. To study mechanisms of resistance to alkylating agents, we have derived an in vitro subline of the murine KHT-iv sarcoma, KHT-rcp/iv, that has acquired resistance to cyclophosphamide. Compared to the parental line, KHT-rcp/iv exhibited an approximately 2.8-fold resistance, determined from the ratio of the slopes of the resistant to the parental survival curve. The subline also was found to be cross-resistant to other alkylating agents, as well as to the epipodophyllotoxin, etoposide, a structurally unrelated compound (resistance factor of approximately 2.2). In view of this broad spectrum of cross-resistance observed, we investigated the possibility that glutathione (GSH), an abundant tripeptide implicated in the detoxification of electrophilic species, may be involved. GSH levels were increased two-fold in the resistant line (16.0 +/- 1.0 fmol/cell) as compared to the parent line (8.1 +/- 0.7 fmol/cell). Also, when the GSH content was reduced to that of the parent with the addition of the thiol depletor buthionine sulfoxinine (BSO), the sensitivity of the subline to cyclophosphamide, as well as to the other agents, was enhanced so that it equaled that of the parent cell line. These results imply that enhanced GSH levels may be a major mechanism of resistance in the KHT-rcp/iv cells. Our findings further suggest that thiol manipulation may prove useful in overcoming this type of pleiotropic drug resistance.

The effect of etoposide (VP-16) on a human epidermoid carcinoma cell line (HEp3) has been evaluated under different growth conditions. A significant increase in resistance to this agent from exponential to unfed plateau cells has been observed. Variations in the cell cycle distribution appear not to be sufficient to explain the difference in cell killing. Rather, our studies suggest the presence of an important proportion of quiescent (Q) cells in the plateau phase cultures which may determine the treatment outcome.

PURPOSE: Tumors encounter endoplasmic reticulum stress during tumor growth and activate an adaptive pathway known as the unfolded protein response (UPR). Because this pathway is induced by the tumor microenvironment, it is a promising target for cancer therapy. We have previously demonstrated that X-box binding protein 1 (XBP-1), a key regulator of the UPR, was required for survival under hypoxia and critical for tumor growth in tumor xenografts. In this study, we investigated the role of XBP-1 in regulating tumor angiogenesis. METHODS: We used an intradermal angiogenesis model to quantify the effect of XBP-1 on angiogenesis. We also used a human tumor xenograft model to assay for tumor growth delay. We determined vascular endothelial growth factor (VEGF) expression by quantitative polymerase chain reaction and ELISA. Finally, we stained human pancreatic adenocarcinoma specimens for XBP-1 expression and correlated the expression pattern of XBP-1 with CD31 (endothelial cell marker) expression. RESULTS: We demonstrated that XBP-1 is essential for angiogenesis during early tumor growth. Inhibiting XBP-1 expression by short-hairpin RNA sequence specific for XBP-1 reduced blood vessel formation in tumors from mouse embryonic fibroblast cells and human fibrosarcoma tumor cells (HT1080). Expressing a dominant-negative form of IRE1α also reduced blood vessel formation in tumors. Moreover, expression of spliced XBP-1 (XBP-1s) restored angiogenesis in IRE1α dominant-negative expressing cells. We further demonstrated that XBP-1-mediated angiogenesis does not depend on VEGF. CONCLUSIONS: We propose that the IRE1α-XBP-1 branch of the UPR modulates a complex proangiogenic, VEGF-independent response that depends on signals received from the tumor microenvironment.

This study examines the effect of irradiation on lung surfactant synthesis and secretion in mice. Animals were irradiated with 650, 1300, or 1950 rad and morphological and biochemical indices of surfactant system function were followed for 18 weeks. No changes were seen at 650 rad; the results at 1300 and 1950 rad were virtually identical. Increased amounts of alveolar surfactant phospholipid were measureable by 24 hours. This persisted for four weeks and returned to normal by 18 weeks. Tissue surfactant phospholipid was initially reduced, returned to normal by four weeks and was increased at 18 weeks. At 18 weeks there was increased incorporation of surfactant precursor and increased production of alveolar surfactant. These biochemical changes were reflected in morphologic alterations showing release of lamellar body contents into alveoli in the first week and an increase in Iamellar bodies in type 11 pneumocytes by 18 weeks. Elevated tissue protein levels and morphologic evidence of increased collagen formation were also found at 18 weeks. These findings indicate effects of irradiation on the pulmonary surfactant system and have important implications for the pathogenesis and potential therapy of radiation pneumonitis.

Surfactant precursors or other products of Type Il pneumocytes have the potential to be the first biochemical marker for late radiation effects. This is particularly clinically important in the combined modality era because of the frequent occurrence of pneumonitis and pulmonary fibrosis secondary to radiation or chemotherapy. Accordingly, correlative studies have been pursued with the Type II pneumocyte as a beginning point to understand the complex pathophysiology of radiation pneumonitis and fibrosis. From our ultrastructural and biochemical studies, it is evident that Type lI pneumocytes are an early target of radiation and the release of surfactant into the alveolus shortly after exposure persists for days and weeks. Through the use of lavaging techniques, alveolar surfactant has been elevated after pulmonary irradiation. In three murine strains and in the rabbit, there is a strong correlation with surfactant release at 7 and/or 28 days in vivo with later lethality in months. In vitro studies using cultures of type lI pneumocytes also demonstrate dose response and tolerance factors that are comparable to the in vivo small and large animal diagnostic models. New markers are being developed to serve as a predictive index for later lethal pneumonopathies. With the development of these techniques, the search for early biochemical markers in man have been undertaken. Through the use of biochemical, histological, and ultrastructural techniques, a causal relationship between radiation effects on type II pneumocytes, pulmonary cells, endothelial cells of blood vessels, and their roles in the production of pneumonitis and fibrosis will evolve.

The lungs of 3, 4, 6 or 12 month old mice were locally irradiated with a single dose of 1100 rad and the time of animal death was determined. The findings indicate that the onset of the animal deaths is a function of the age of the animals at the time of treatment; the older mice were more resistant to radiation. The median time of death was 20 weeks (134–140 days) for 3 and 4 month old mice, 24 weeks (162–168 days) for 6 month old mice, and 28 weeks (190–196 days) for 12 month old mice.

Studies were performed to determine whether the radiation sensitizer misonidazole (MISO) could enhance the tumor control probability in a treatment strategy combining radiation and the nitrosourea 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). In initial experiments KHT sarcoma-bearing mice were injected with 1.0 mg/g of MISO simultaneously with a 20 mg/kg dose of CCNU 30-40 min prior to irradiation (1500 rad). These timings were chosen to maximize the effectiveness of MISO both as a chemopotentiator and radiosensitizer. With this treatment protocol approximately 60% of the mice were found to be tumor-free 100 days post treatment. By comparison all 2 agent combinations led to 0% cures. To evaluate the relative importance of chemopotentiation versus radiosensitization in the 3 agent protocol, tumors were treated with MISO plus one anti-tumor agent (either radiation of CCNU) and then at times ranging from 0 to 24 hr later exposed to the other agent. When the time between treatments was 0 to 6 hr, a 60 to 80% tumor control rate was achieved for both MISO plus radiation followed by CCNU and MISO plus CCNU followed by radiation. However if the time interval was increased to 18 or 24 hr, the cure rate in the former treatment regimen dropped to 10% while that of the latter remained high at 40%. These results were not due to the radiation-CCNU sequence but rather reflected the ability of the sensitizer to act as a chemopotentiator when CCNU is given 0 to 6 hr after the MISO-radiation combination. This was not the case when the MISO-radiation combination was administered 18 or 24 hr prior to CCNU. The data therefore indicate that 1) improved tumor responses may be achieved when MISO is added to a radiation-chemotherapy combination and 2) MISO may be more effective in such a protocol when utilized as a chemopotentiator.

The radiation sensitizer misonidazole (MISO) has been shown to potentiate the cytotoxic action of a variety of anti-cancer agents. Even larger enhancement ratios than those observed with MISO have been found with certain other nitroimidazoles. One agent reported to be particularly effective in combination with the chemotherapeutic agent melphalan is the sensitizer RSU 1069. The present studies therefore were designed to evaluate the effect of combining these two agents in the treatment of intramuscularly growing KHT sarcomas. Tumor response was assessed using an in vivo to in vitro clonogenic cell survival assay. When given at times ranging from 60 min before to 30 min after melphalan exposure, RSU 1069 was found to increase the tumoricidal activity of the chemotherapeutic agent. Complete dose response curves combining RSU 1069 and a range of melphalan doses then were determined. For comparison the effects of combining MISO or benznidazole (BENZO) with melphalan also were evaluated. All sensitizers were administered i.p. either 30 min before (BENZO) or simultaneously with (MISO, RSU 1069) the chemotherapeutic agent. Survival of clonogenic tumor cells assessed 22 to 24 hr after treatment was used to assay tumor response. When combined with melphalan, doses of RSU 1069 (0.38 mmol/kg), BENZO (0.3 mmol/kg) and MISO (5.0 mmol/kg) were found to yield dose modifying factors of 1.6, 1.5, and 1.4, respectively. These results indicate that potentiation of melphalan activity occurs at RSU 1069 doses which are approximately 10-fold lower than those of MISO, making this sensitizer as effective a potentiator of melphalan as so far tested in the KHT sarcoma.

Tumor response to radiation is dependent not only on the quantity of hemoglobin (Hb) available for oxygen (O2) transport but also on the position of the Hb-O2 dissociation curve (Hb affinity). Previous studies have shown that administering agents which shift the Hb-O2 dissociation curve to the right (decrease Hb affinity) sensitize tumors to radiation by reducing the fraction of radiobiologically hypoxic cells. However, there may be toxicity limitations when agents aimed at altering Hb affinity are administered directly to the host. The present studies evaluated the therapeutic benefit of shifting the Hb-O2 dissociation curve in vitro prior to the transfusion of the biochemically modified RBCs into recipient hosts. Mice were given a hemolysis agent (phenylhydrazine hydrochloride, PH) prior to transfusing RBCs with normal or altered Hb affinity. A 100 mg/kg dose of PH reduced the hematocrit to approximately 60% of control 24 hr after treatment. Tumors irradiated at this time demonstrated an increased fraction of hypoxic cells. If the hematocrit was returned to normal by transfusing mice prior to irradiation, a significant but transient reduction in the hypoxic fraction was seen. Tumor response was reduced if RBCs with elevated Hb affinity, obtained by storing the erythrocytes at 4 degrees C, were used. Alternatively, tumor sensitization was noted when animals were transfused with RBCs having decreased Hb affinities. The latter was achieved by incubating the RBCs in the presence of either clofibrate or the precursors of 2,3 diphosphoglycerate (2,3 DPG). These findings further support the notion that the Hb affinity is an important parameter in determining tumor response to radiation and suggest that this factor ought to be considered when RBCs are used to transfuse anemic patients undergoing radiotherapy.

The pathogenesis of pneumonitis and fibrosis secondary to lung irradiation is incompletly understood. The role of the type II alveolar epithelial pneumocyte in these processes has been under investigation. The type II pneumocyte has been shown in vivo to respond to radiation induced injury with release of pulmonary surfactant. The effect of irradiation on cell cultures of type II pneumocytes was studied to determine if this could be reproduced in vitro . Type II pneumocytes were found to release surfactant material with a threshold of radiation dose between 1000 and 1500 rad. This is similar to the dosage range over which the same effect has been demonstrated in vivo . Experimental results support the concept that the release of surfactant is not due to either cell disruption or non-specific release of phospholipid from cell membranes. Irradiation appears to trigger membrane receptor mediated surfactant release. In addition, irradiation abolishes the ability of cells to subsequently respond to a physiologic agonist, suggesting radiation induced damage to the secretory mechanism. These studies establish that surfactant release in response to irradiation in vivo is a direct effect on type II pneumocytes. Cell cultures of type II pneumocytes can serve as a laboratory model of lung cell radiation toxicity.

The effect of single or fractionated doses of radiaton on the transplantable KHT sarcoma has been studied in mice which were exposed to a reduced oxygen (O 2 ) environment prior to but not during irradiation. Tumor-bearing mice were kept in a 12% O 2 environment for a period of time; then their tumors were irradiated locally with a single dose of 2000 rad at a dose rate of 1140 rad/min while the mice breathed air, O 2 or carbogen (5% CO 2 :95% O 2 ). Following irradiation, tumor cell survival was determined using an in vivo lung colony or an in vitro agar colony assay. The results indicate that overnight exposure of tumor-bearing mice (15 hr) to 12% O 2 , prior to irradiation of their tumors under air-breathing conditions, leads to a reduction in tumor cell survival by a factor of 3–4 over that found for tumors of air-breathing mice not given a low O 2 pre-treatment. A similar reduction in tumor cell survival is observed for irradiation without prior exposure to the low O 2 environment when the mice are given O 2 or carbogen to breathe instead of air. If the tumors are irradiated while the animals breathe O 2 or carbogen after exposure to 12% O 2 , tumor cell survival is reduced to a value ∼10 times lower than is obtained in tumors of air-breathing mice not given a low O 2 pre-treatment. During a course of fractionated radiation (seven 500 rad fractions at 24 hr intervals) delivered while the tumor-bearing mice breathed air or carbogen, a 12% O 2 exposure prior to alternate dose fractions was found to lead to significant enhancement of tumor cell killing above that observed when no pre-treatment with 12% O 2 was employed. The reduction in tumor cell survival almost certainly results from a decrease in the number of hypoxic cells in the tumors but the mechanism by which this reduction occurs is not known at present. The decrease may be because of direct killing of hypoxic cells by the low O 2 environment or because of physiological adaptation to the low O 2 environment which persists when the animals are returned to an air atmosphere, leading to an improved O 2 delivery to the tumor. A number of adaptation mechanisms were studied but these shpwed little correlation with the radiobiological effect. Consequently, no clear explanation of the results can be given at present. Regardless of the mechanism, the findings indicate an approach to increasing the effectiveness of radiation treatment of tumors.

A spontaneously originated murine mammary adenocarcinoma (16C), selected for its sensitivity to agents active against breast cancer in women, and one of the very few experimental solid tumor models responsive to Adriamycin (ADR) was used to study the mechanism of induced ADR resistance in vivo. A resistant variant of the tumor was obtained from the explant of a regrown tumor following a dose of ADR (12 mg/kg) that caused complete tumor repression but not cure. Progressive refractoriness to ADR was observed following up to six repeated cycles of treatment, regression and regrowth. However, beyond the sixth treatment, no further degree of resistance could be obtained. The cell line so established, designated 16C/ADRR, has a glutathione (GSH) content 1.67 times greater than the parent 16C line. Depletion of GSH by buthionine sulfoximine (BSO) enhanced the cytoxicity of ADR in both cell lines. The sensitization effect appeared to be dependent on the degree of GSH depletion, requiring a threshold level of depletion to approximately 30% of control. The resistance of 16C/ADRR, however, appeared not to be directly related to the increased absolute GSH level per se since reduction of the GSH content of the 16C/ADRR line to levels similar to that of the parent 16C line did not restore the original sensitivity to ADR. However, the activities of two important elements in the GSH detoxification system, GSH peroxidase and S-transferase, were found to be elevated in resistant cells by factors of 2.4 and 4.7-5.6 respectively. In vivo studies with a diverse spectrum of antineoplastic drugs revealed a pattern of cross-resistance consistent with the idea that elevated GSH S-transferase and peroxidase activities may be responsible for the decreased (2.8- to 5.3-fold) sensitivity to ADR. 16C/ADRR exhibited cross-resistance with melphalan (MEL), but none with vincristine (VCR), vinblastine (VBL) or etoposide (VP-16). These results clearly demonstrate non-adherence by the 16C/ADRR tumors to the well characterized multidrug resistance (mdr) phenotype. Further affirmation of this conclusion was obtained by immunochemical and pharmacological studies. When a monoclonal antibody prepared against the mdr associated, 170 kD P-glycoprotein (170 P-gp), was used, the presence of the 170 kD P-gp in both the sensitive and resistant 16C lines could not be detected, although the presence of a lower molecular weight form of P-gp could not be ruled out entirely. High performance liquid chromatographic measurement of ADR accumulation and elimination also failed to reveal any significant differences between the sensitive and resistant variants.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

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.

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.