Overexpression of VEGF121, but not VEGF165 or FGF-1, improves oxygenation in MCF-7 breast tumours.

Vascular endothelial growth factor (VEGF) is an intensively studied molecule that has significant potential, both in stimulating angiogenesis and as a target for antiangiogenic approaches. We utilised MCF-7 breast cancer cells transfected with either of two of the major VEGF isoforms, VEGF(121) or VEGF(165), or fibroblast growth factor-1 (FGF-1) to distinguish the effects of these factors on tumour growth, vascular function, and oxygen delivery. While each transfectant demonstrated substantially increased tumorigenicity and growth rate compared to vector controls, only VEGF(121) produced a combination of significantly reduced total and perfused vessel spacing, as well as a corresponding reduction in overall tumour hypoxia. Such pathophysiological effects are of potential importance, since antiangiogenic agents designed to block VEGF isoforms could in turn result in the development of therapeutically unfavourable environments. If antiangiogenic agents are also combined with conventional therapies such as irradiation or chemotherapy, microregional deficiencies in oxygenation could play a key role in ultimate therapeutic success.

Mechanism of IL-12 mediated alterations in tumour blood vessel morphology: analysis using whole-tissue mounts.

New blood vessel formation within tumours is a critical feature for tumour growth. A major limitation in understanding this complex process has been the inability to visualise and analyse vessel formation. Here, we report on the development of a whole-tissue mount technique that allows visualisation of vessel structure. Mice expressing green fluorescent protein (GFP) made it possible to easily see GFP(+) vessels within non-GFP-expressing B16 melanoma tumours. The small fragments of tumour used in this technique were also effectively stained with fluorescent probe-conjugated antibodies, allowing characterisation of the vessels based on surface marker phenotype. The vessels within tumour tissue were much more irregular and tortuous compared to those within surrounding normal muscle. B16 tumours stably transfected with the genes for IL-12 were used to assess the effects of this cytokine on tumour growth and vessel formation. The IL-12-expressing tumours grew more slowly and had much smaller blood vessels than the large, webbed vessels characteristic of the parental tumours, effects that were dependent on interferon gamma (IFN-gamma). Vessels in the parental tumours were found to express VEGFR-3, the receptor for VEGF-C and VEGF-D. Expression of this receptor by the endothelial cells of the blood vessels was lost in the cytokine expressing tumours, thus suggesting a mechanism for the antiangiogenic effects of IL-12. The combination of the whole mount technique and the GFP transgenic mice provides a powerful method for visualising tumour vasculature and characterising the effects of agents such as cytokines.

Zonal image analysis of tumour vascular perfusion, hypoxia, and necrosis.

A number of laboratories are utilising both hypoxia and perfusion markers to spatially quantify tumour oxygenation and vascular distributions, and scientists are increasingly turning to automated image analysis methods to quantify such interrelationships. In these studies, the presence of regions of necrosis in the immunohistochemical sections remains a potentially significant source of error. In the present work, frozen MCa-4 mammary tumour sections were used to obtain a series of corresponding image montages. Total vessels were identified using CD31 staining, perfused vessels by DiOC(7) staining, hypoxia by EF5/Cy3 uptake, and necrosis by haematoxylin and eosin staining. Our goal was to utilise image analysis techniques to spatially quantitate hypoxic marker binding as a function of distance from the nearest blood vessel. Several refinements to previous imaging methods are described: (1) hypoxia marker images are quantified in terms of their intensity levels, thus providing an analysis of the gradients in hypoxia with increasing distances from blood vessels, (2) zonal imaging masks are derived, which permit spatial sampling of images at precisely defined distances from blood vessels, as well as the omission of necrotic artifacts, (3) thresholding techniques are applied to omit holes in the tissue sections, and (4) distance mapping is utilised to define vascular spacing.

Characterization of the effects of antiangiogenic agents on tumor pathophysiology.

A variety of strategies have been proposed to control tumor growth and metastasis by inhibiting tumor angiogenesis. To optimally combine such antiangiogenic approaches with conventional therapy, improved methods are needed to characterize the underlying pathophysiologic changes. The objective of the current work was to demonstrate the utility of a combination of recently developed immunohistochemical and image analysis techniques in quantitating changes in tumor vasculature and hypoxia. Murine MCa-35 mammary carcinomas were frozen after administration of two COX-2 inhibitors: meloxicam and celecoxib (Celebrex). Total blood vessels were visualized using anti-CD31 staining, perfused vessels by intravenous injection of DiOC7, and tumor hypoxia by EF5 uptake. Although both agents produced similar reductions in tumor volume compared with untreated tumors, varied effects on tumor vasculature and hypoxia were noted. Meloxicam reduced total vessel numbers significantly, whereas celecoxib had no effect. Both drugs substantially increased perfused vessel densities. Although mean hypoxic marker uptake was unchanged from matched controls, intratumor EF5 heterogeneities were significantly different between drugs. The results suggest that COX-2 inhibitors can have varying effects on tumor pathophysiology. Successful use of these drugs to enhance radiation response will likely require optimization of drug choice, dose schedule, and direct physiologic monitoring.

Intravascular HBO(2) saturations, perfusion and hypoxia in spontaneous and transplanted tumor models.

Clinical trials utilizing strategies to manipulate tumor oxygenation, blood flow and angiogenesis are under way, although limited quantitative information exists regarding basic tumor pathophysiology. The current study utilized murine KHT fibrosarcomas, spontaneous mammary carcinomas and first-generation spontaneous transplants to examine heterogeneity in vascular structure and function, to relate these changes to the distribution of tumor hypoxia and to determine whether fundamental relationships among the different pathophysiological parameters exist. Three methods were included: (i) immunohistochemical staining of anatomical and perfused blood vessels, (ii) cryospectrophotometric measurement of intravascular oxyhemoglobin saturations and (iii) fluorescent detection of the EF5 hypoxic marker. While a distinct pattern of decreasing oxygenation with increasing distance from the tumor surface was observed for KHT tumors, striking intertumor variability was found in both spontaneous and first-generation transplants, with a reduced dependence on tumor volume. EF5 hypoxic marker uptake was also much more heterogeneous among individual spontaneous and first-generation tumors compared to KHT. Although mammary carcinomas demonstrated fewer anatomical blood vessels than fibrosarcomas, the proportion of perfused vessels was substantially reduced in KHT tumors, especially at larger tumor volumes. Vascular morphology, tissue histological appearance and pathophysiological parameters differed substantially between KHT tumors and both spontaneous and first-generation tumors. Such differences in vascular structure and function are also likely to correlate with altered response to therapies targeted to the vascular system. Finally, spontaneous differentiation status, tumor morphology, vascular configuration and function were well preserved in first-generation transplanted tumors, suggesting a close relationship between vascular development and function in early-generation transplants and spontaneous tumor models.

Influence of hydralazine administration on oxygenation in spontaneous and transplanted tumor models.

PURPOSE: To examine the effects of hydralazine on vascular perfusion and hypoxia in spontaneous vs. first generation and long-term transplanted murine tumor models. METHODS AND MATERIALS: Total anatomic blood vessels were quantified using image analysis of CD31 stained frozen sections, perfused vessels by i.v. injection of fluorescent DiOC(7), and tumor hypoxia was measured using the EF5 hypoxia marker. KHT sarcomas, spontaneous mammary carcinomas, and first generation transplants of the spontaneous tumors were evaluated before and after i.p. administration of 5 mg/kg hydralazine. RESULTS: Although anatomic and perfused vessel spacings were similar among untreated tumors, response to hydralazine varied widely among the three tumor models. In KHT tumors, perfused vessel numbers decreased significantly at 30 min post-hydralazine, then recovered somewhat by 60 min. First-generation transplants showed a less substantial decrease in perfused vessels following hydralazine, which tapered off slightly by 60 min. Finally, spontaneous tumors had only a modest decrease in perfused vessel numbers, with complete recovery at 60 min. Although response of individual tumors varied widely, overall hypoxic marker uptake was significantly increased in both KHT and first generation tumors, and slightly reduced in the spontaneous tumors. CONCLUSION: Response to hydralazine varies substantially between transplanted and spontaneous tumor models. Results suggest that increased tumor pressure may be a critical factor in tumor response to hydralazine, possibly explaining tumor volume dependent variations.

Effects of radiation on tumor intravascular oxygenation, vascular configuration, development of hypoxia, and clonogenic survival.

The underlying physiological mechanisms leading to tumor reoxygenation after irradiation have elicited considerable interest, but they remain somewhat unclear. The current study was undertaken to determine the effects of a single dose of 10 Gy gamma radiation on both tumor pathophysiology and radiobiologically hypoxic fraction. Immunohistochemical staining and perfusion markers were used to quantify tumor vasculature, uptake of the hypoxia marker EF5 to assess the distribution of hypoxia, and intravascular HbO(2) measurements to determine oxygen availability. Tumor radiosensitivity was measured by a clonogenic assay. At 24 h postirradiation, oxygen availability increased, perfused vessel numbers decreased, EF5 uptake decreased, and the radiobiologically hypoxic fraction was unchanged. Together, these results demonstrate that tumor hypoxia develops at an increased distance from perfused blood vessels after irradiation, suggesting a decrease in oxygen consumption at 24 h. By 72 h postirradiation, all physiological parameters had returned to the levels in volume-matched, nonirradiated controls. These studies clearly show that single measures of either tumor oxygenation or vascular structure are inadequate for assessing the effects of radiation on tumor clonogenicity. Although such direct measurements have previously proven valuable in predicting tumor response to therapy or oxygen manipulation, a combination of parameters is required to adequately describe the mechanisms underlying these changes after irradiation.

An evaluation of near infrared spectroscopy and cryospectrophotometry estimates of haemoglobin oxygen saturation in a rodent mammary tumour model.

Haemoglobin oxygen saturation in subcutaneous rat mammary tumours was measured using near infrared spectroscopy (NIRS) in vivo and in rapidly frozen sections from the same tumours using cryospectrophotometry, which reports oxygen saturation in individual blood vessels to depths of 4 mm from the tissue surface. Measurements were performed on tumours while animals breathed either room air or carbogen. In five of nine tumours, the average saturation calculated from cryospectrophotometric measurements agreed with that determined from NIRS to within 13%, and in four of these five tumours agreement was 5% or better. In the remaining four of nine tumours, where agreement was poor, the volume-averaged saturations estimated from NIRS were consistently higher than those calculated from cryospectrophotometry. Monte Carlo simulations demonstrated that the depth of tissue probed by NIRS was significantly greater than that sampled by cryospectrophotometry. Analysis of the frequency of severely hypoxic vessels showed that when NIRS reported a saturation of approximately 70% or higher, the fraction of tumour vessels with saturations less than 10% was limited to 0.06 or less. Sensitivity and specificity analysis suggests that NIRS and NIRS imaging may identify clinically relevant hypoxia, even when its spatial extent is below the resolution limit of the NIRS technique.

Alteration of tumour response to radiation by interleukin-2 gene transfer.

We have previously shown that BALB/c-derived EMT6 mammary tumours transfected with interleukin (IL)-2 have decreased hypoxia compared to parental tumours, due to increased vascularization. Since hypoxia is a critical factor in the response of tumours to radiation treatment, we compared the radiation response of IL-2-transfected tumours to that of parental EMT6 tumours. Because the IL-2 tumours have an altered host cell composition, which could affect the interpretation of radiation sensitivity as measured by clonogenic cells, we employed flow cytometric analysis to determine the proportion of tumour cells vs host cells in each tumour type. Using this approach, we were able to correct the plating efficiency based on the number of actual tumour cells derived from tumours, making the comparison of the two types of tumours possible. We also excluded the possibility that cytotoxic T-cells present in EMT6/IL-2 tumours could influence the outcome of the clonogenic cell survival assay, by demonstrating that the plating efficiency of cells derived from EMT6/IL-2 tumours remained unchanged after depletion of Thy-1+ cells. The in vivo radiation response results demonstrated that IL-2-transfected tumours were more sensitive to radiation than parental EMT6 tumours. The hypoxic fraction of the EMT6/IL-2 tumours growing in vivo was markedly decreased relative to parental EMT6 tumours thus the increased sensitivity results from the increased vascularity we have previously observed in these tumours. These results indicate the potential therapeutic benefit of combining radiation and immunotherapy in the treatment of tumours.

Enhancement of tumor perfusion and oxygenation by carbogen and nicotinamide during single- and multifraction irradiation.

Numerous experimental and clinical studies have been completed regarding the effects of carbogen and nicotinamide on tumor oxygenation and radiosensitivity. The current study incorporates three physiological measurement techniques to further define spatial variations in oxygen availability and development of hypoxia after single- and multifraction irradiation in KHT murine fibrosarcomas. Distances to anatomical and perfused blood vessels were measured using immunohistochemical and fluorescent staining, intravascular oxygen levels were determined cryospectrophotometrically, and tumor hypoxia was quantified using uptake of EF5, a marker of hypoxia. Carbogen, nicotinamide, and the combination of both all increased intravascular oxygen availability compared to controls. While nicotinamide had no effect on the number of perfused blood vessels in nonirradiated tumors, carbogen produced a substantial closing of vessels. After a single dose of 4 Gy, only the combination of nicotinamide and carbogen produced significant improvements in oxygen availability, while numbers of perfused vessels were significantly increased for nicotinamide, unchanged for the combination of nicotinamide and carbogen, and significantly decreased for carbogen. After 4 x 4-Gy fractions, oxygen availability was increased substantially with the combination of nicotinamide and carbogen, somewhat with carbogen, and not at all with nicotinamide. Tumor oxygenation changes were estimated by EF5/Cy3 intensity distributions, which demonstrated that manipulative agents could produce disparate effects on tumor hypoxia when combined with either single- or multifraction irradiation.

Quantification of tumour vasculature and hypoxia by immunohistochemical staining and HbO2 saturation measurements.

Despite the possibility that tumour hypoxia may limit radiotherapeutic response, the underlying mechanisms remain poorly understood. A new methodology has been developed in which information from several sophisticated techniques is combined and analysed at a microregional level. First, tumour oxygen availability is spatially defined by measuring intravascular blood oxygen saturations (HbO2) cryospectrophotometrically in frozen tumour blocks. Second, hypoxic development is quantified in adjacent sections using immunohistochemical detection of a fluorescently conjugated monoclonal antibody (ELK3-51) to a nitroheterocyclic hypoxia marker (EF5), thereby providing information relating to both the oxygen consumption rates and the effective oxygen diffusion distances. Third, a combination of fluorescent (Hoechst 33342 or DiOC7(3)) and immunohistological (PECAM-1/CD31) stains is used to define the anatomical vascular densities and the fraction of blood vessels containing flow. Using a computer-interfaced microscope stage, image analysis software and a 3-CCD colour video camera, multiple images are digitized, combined to form a photo-montage and revisited after each of the three staining protocols. By applying image registration techniques, the spatial distribution of HbO2 saturations is matched to corresponding hypoxic marker intensities in adjacent sections. This permits vascular configuration to be related to oxygen availability and allows the hypoxic marker intensities to be quantitated in situ.

Characterization of the dose perturbation by stents as a function of X-ray beam energy.

PURPOSE: External beam irradiation of coronary arteries has been shown to be detrimental in an animal model for the prevention of neointimal hyperplasia in the presence of stents when orthovoltage x-ray beams are used. The present study investigated the effect of beam energy on the dose distribution in the wall of the artery in the presence of stents. MATERIALS AND METHODS: We used 250-kVp x-rays and 6-MV x-rays to irradiate a stent placed in a homogeneous phantom. Radiochromic film densitometry and Monte Carlo calculations were used to measure and to simulate the dose distribution in the proximity of the stent. RESULT: External beam irradiation not only failed to prevent neointimal hyperplasia, but actually accentuated the neointimal response to a prompt mechanical injury in the artery. The photoelectric effect, which dominates low-energy x-ray interactions, produces recoil electrons in the stent, which enhance the dose surrounding the intima. The photoelectrons generated in nickel and iron have an extremely short range in normal tissue, approximately 0.1 mm. Initial estimates of orthovoltage x-ray interactions with the stent indicate a dose enhancement in the orthovoltage range by a factor of 2-6 due to the rise in the photoelectric cross section in this energy range depending on the elemental composition of the stent. Film densitometry verifies this dose enhancement. The Monte Carlo calculation yields a dose enhancement and the dose fall-off with distance from the stent when irradiated with orthovoltage x-rays. Conversely when the tissue and stent are irradiated with megavoltage x-rays, the dose enhancement in this region is a factor of 1.15 in close proximity to the stent and 1.0 at distances greater than 0.1 mm. The 6-MV photon interactions in tissue and Ni/Ti are predominantly through Compton scattering. The Compton effect is dependent on the electron density in the medium, in contrast to the atomic number, which is more relevant for photoelectric absorption. The dose estimates for megavoltage x-rays adjacent to the stent are complicated by the lack of charged particle equilibrium. CONCLUSIONS: There is a limited but definite increase in the dose delivery to the arterial wall when stents are irradiated with orthovoltage x-ray energies. This increase may explain the negative response in other studies. The presence of the stent does perturb the character and magnitude of the dose in the normal arterial wall as a function of beam quality.

Interleukin 2 expression by tumor cells alters both the immune response and the tumor microenvironment.

Microenvironmental conditions within solid tumors can have marked effects on the growth of the tumors and their response to therapies. The disorganized growth of tumors and their attendant vascular systems tends to result in areas of the tumors that are deficient in oxygen (hypoxic). Cells within these hypoxic areas are more resistant to conventional therapies such as radiation and chemotherapy. Here, we examine the hypoxic state of EMT6 mouse mammary tumors and the location of host cells within the different areas of the tumors to determine whether such microenvironmental conditions might also affect their ability to be recognized by the immune system. Hypoxia within tumors was quantified by flow cytometry and visualized by immunohistochemistry using a monoclonal antibody (ELK3-51) against cellular adducts of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetam ide (EF5), a nitroimidazole compound that binds selectively to hypoxic cells. Thy-1+ cells, quantified using a monoclonal antibody, were found only in the well-oxygenated areas. The location of these Thy-1+ cells was also examined in EMT6 tumors that had been transfected with the gene for interleukin-2 (IL-2) because these tumors contain greatly increased numbers of host cells. Surprisingly, we found that IL-2-transfected tumors had significantly decreased hypoxia compared to parental tumors. Furthermore, using the fluorescent dye Hoechst 33342, an in vivo marker of perfused vessels, combined with immunochemical staining of PECAM-1 (CD31) as a marker of tumor vasculature, we found increased vascularization in the IL-2-transfected tumors. Thus, expression of IL-2 at the site of tumor growth may enhance tumor immunity not only by inducing the generation of tumor-reactive CTLs but also by allowing increased infiltration of activated T cells into the tumors.

Effects of carbogen plus fractionated irradiation on KHT tumor oxygenation.

BACKGROUND AND PURPOSE: Numerous studies have demonstrated improvements in the oxygenation of tumor cells following both irradiation and carbogen breathing. The current studies were initiated to measure the combined effects of carbogen inhalation plus single and multi-dose irradiation on tumor oxygen availability, to better define the underlying physiological relationships. MATERIALS AND METHODS: Using KHT murine sarcomas, radiation was delivered to the tumor-bearing legs of non-anesthetized mice. Tumors were quick-frozen prior to or following single or multifraction irradiation and carbogen breathing, and intravascular HbO2 saturation profiles were determined cryospectrophotometrically. RESULTS: HbO2 levels for blood vessels located near the tumor surface initially decreased following 10 Gy irradiation, then increased and remained elevated. Interior HbO2 levels remained unchanged. Following 2.5 Gy, HbO2 changes were minimal. At 24 h following 10 Gy, HbO2 levels were significantly increased compared to non-irradiated controls, and carbogen breathing produced no additional benefit. At 24 h following five fractions of 2 Gy, HbO2 levels throughout the tumor volume were significantly higher in carbogen breathing animals than in air breathing controls. CONCLUSIONS: Although peripheral blood vessels demonstrated substantial improvements in oxygenation following irradiation, oxygen availability nearer the tumor center remained at very low levels. The utility of carbogen in enhancing tumor oxygen availability was maintained following five clinically relevant fractions. At higher doses, radiation-induced enhancements in HbO2 levels overshadowed the carbogen effect. For either air or carbogen breathing, a decrease in the percentage of vessels with very low oxygen content did not appear to be a major factor in the reoxygenation of the KHT tumor.

Effects of ionizing radiation on the adhesive interaction of human tumor and endothelial cells in vitro.

A centrifugation assay was used to determine the effects of ionizing radiation on the adhesive interaction of A549 human lung adenocarcinoma tumor cells and human umbilical vein endothelial cells (HUVEC). The tumor cells were fluorescently labeled and divided into control (sham-irradiated) and irradiated groups. The irradiated groups were exposed to irradiation levels ranging from 5 to 20 Gy using a 137Cs source. A specified number of these A549 tumor cells were then delivered into each well of 96-well cell culture plates containing confluent monolayers of human umbilical cord vein endothelial cells (HUVEC), and were given time to adhere to the endothelial cells. The wells were then sealed and were exposed to an acceleration field varying from 1 to 42 g (0-500 rpm) for 10 min. Finally, the wells were drained, and the number of tumor cells adhering to the endothelial monolayer were counted using a fluorescent microscope system. Our results indicate that the irradiation of A549 tumor cells significantly increased their adhesive interaction with endothelial cells (number of adhering irradiated cells/number of adhering control cells = 1.0, 1.3, 1.9, 2.2 for 0, 5, 10, 20 Gy respectively). In contrast, when endothelial cells were irradiated, rather than tumor cells, adhesive interaction decreased with an increase in the radiation dose (irradiated/control = 1.0, 0.9, 0. 8, 0.5 for 0, 5, 10, 20 Gy respectively). Simultaneous irradiation of both the tumor cells and the endothelial cells did not alter their adhesive interaction significantly. These findings may have important implications for the metastatic ability of irradiated tumor cells.

Should direct measurements of tumor oxygenation relate to the radiobiological hypoxic fraction of a tumor?

PURPOSE: Numerous previous studies have attempted to relate the radiobiological hypoxic fraction (HF) to direct measures of tumor oxygenation such as HbO2 saturations, tumor pO2 levels, or hypoxic cell labeling. Although correlations have been found within tumor lines, no overall relationships were seen across tumor lines. The current objective was to examine the effect on HF of changes in the fractions of the oxygenated and anoxic tumor cells that remain clonogenic. METHODS AND MATERIALS: A mathematical model was developed that relates the HF to direct measures of tumor oxygenation. The primary assumptions were that: (a) the tumor is divided into distinct compartments of either fully oxygenated or fully anoxic cells, and (b) the survival of the oxygenated cells is negligible compared to that of the anoxic cells. Based on these assumptions, the HF is plotted as a function of the fractions of clonogenic or nonclonogenic, and oxygenated or anoxic cells. RESULTS: If all cells are clonogenic, then the HF equals the fraction of anoxic cells. If a higher fraction of anoxic than oxygenated cells are nonclonogenic, then the HF will be overestimated by the fraction of the tumor measured to be anoxic using direct measuring techniques. If a higher fraction of the oxygenated than anoxic cells are nonclonogenic, the HF will be underestimated by the fraction of anoxic cells. CONCLUSION: Correlations between the HF and direct measures of tumor oxygenation have been described within tumor lines evaluated under different physiological condition. However, such relationships can be totally unpredictable between different tumors if the fraction of the anoxic cells that is clonogenic varies substantially. Clearly, if tumor anoxia cannot be detected using direct measures, this is an accurate indication that the tumor is well oxygenated. When tumor anoxia is present, however, the conclusions are ambiguous. Even when a small fraction of the tumor is measured as anoxic, direct measures of tumor oxygenation may not be representative of the HF if a substantial proportion of nonclonogenic cells is present.

The effects of carbogen and nicotinamide on intravascular oxyhaemoglobin saturations in SCCVII and KHT murine tumours.

Considerable effort has been focused on devising methods for manipulating tumour oxygenation and thereby improving tumour radiosensitivity. The combination of nicotinamide and carbogen has been proposed to oxygenate both chronically and acutely hypoxic cells in tumours. However, results have varied markedly with both tumour model and measurement technique. The current objectives were (1) to determine whether changes in radiosensitivity following oxygen manipulation correlated with changes in tumour oxygenation and (2) to assess whether oxygenation was preferentially improved in specific tumour micro-regions. Using two murine tumour lines, the SCCVII carcinoma and the KHT sarcoma, tumour intravascular HbO2 saturations were measured cryospectrophotometrically following nicotinamide, carbogen or the combination. Generally, nicotinamide had minor effects on oxygenation, arguing against a substantial effect on acute hypoxia, while carbogen and the combination produced marked and equivalent improvements in oxygen availability. These results demonstrate that changes in tumour radiosensitivity may not agree with corresponding changes in oxygenation, even within a given tumour model, and that the efficacy of a given manipulative agent may vary substantially with tumour line. One possible explanation for these findings is that different subpopulations of clonogenic vs non-clonogenic cells may be oxygenated by alternative treatments.

Inherent cellular differences may explain the dissimilar survival of RIF-1 and KHT tumour cells under aerobic and hypoxic conditions.

Although previous work has shown striking differences in radiobiological hypoxic fraction between KHT and RIF-1 murine sarcomas, intravascular oxyhaemoglobin (HbO2 saturations have revealed less substantial variations. Using quantitative histological techniques, we have also found minor differences in the distributions of distances between tumour cells and the nearest bloods vessel for KHT versus RIF-1 sarcomas. We report here, the results of an investigation of the inherent ability of these tumour cells to withstand conditions of hypoxia by in vitro culturing under aerobic and anoxic conditions. Tumours were dissociated, seeded into culture dishes, and placed in air-tight aluminium chambers. These chambers were repeatedly evacuated and refilled with a mixture of 95% N2 and 5% CO2 over a 2.5-h period. Following anoxic exposure, cells were removed and replated, and the in vitro plating efficiency (PE) was determined using a colony survival assay. After normalizing to aerobic controls, KHT tumour cells had a significantly lower PE, following a 16-hour exposure to anoxic conditions (0.4), than RIF-1 (0.6). Increasing the hypoxic exposure to 40 h resulted in normalized PEs of 0.07 for KHT versus 0.4 for RIF-1. Although these results support the hypothesis that the two tumour lines have different inherent abilities to withstand hypoxia, they do not explain the failure of direct measures of tumour oxygenation to correlate with the radiobiological hypoxic fraction. Additional factors such as differences in oxygen diffusivity or oxygen consumption rates between tumour lines may also be involved.

Quantification of micro-regional heterogeneities in tumor oxygenation using intravascular HbO2 saturations.

Despite promising reports as to the relationship between tumor radiosensitivity and overall tumor oxygenation, little additional information has been forthcoming regarding the importance of localized variations in tumor oxygen distribution. The objectives of the current study were (1) to devise a strategy for sampling tumor HbO2 saturation distributions and (2) to compare intratumor heterogeneities in HbO2 profiles with intertumor variability to determine whether representative tumor oxygen profiles can be obtained from a limited tumor sample. Using murine KHT fibrosarcomas, maps of tumor intravascular HbO2 saturations were obtained using cryospectrophotometric techniques and without the use of anesthetics. Micro-regions composed primarily of either high or low HbO2 vessels were observed in both peripheral and interior regions of the KHT tumors, although HbO2 levels were higher, on average, toward the periphery. To quantify intra- and intertumor heterogeneities in oxygen delivery, alternative HbO2 sampling protocols were evaluated in comparison to all-inclusive HbO2 maps for each tumor cross section. Since intratumor variations in HbO2 distributions were of the same order of magnitude as intertumor variations for tumors of a single tumor line, it is clear that tumor micro-regional physiology cannot be characterized adequately by a single regional sample.