Different activation signals detected by fixation of stimulator cells.
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Animals↗
Biomedical subjects
R M Sutherland
Publications and source records attributed to R M Sutherland.
Cryospectrophotometric determination of tumor intravascular oxyhemoglobin saturations: dependence on vascular geometry and tumor growth.
To delineate the complex relationships between overall tumor oxygenation and vascular configuration, intravascular oxyhemoglobin (HbO2) saturation distributions were measured with cryospectrophotometric techniques. Four factors related to vascular morphometry and tumor growth were evaluated: a) vessel diameter, b) distance of vessel from the tumor surface, c) tumor volume, and d) vascular density. To measure intertumor heterogeneity, two murine sarcomas (RIF-1 and KHT) and two human ovarian carcinoma xenografts (OWI and MLS) were utilized. In contrast to skeletal muscle, a preponderance of very low HbO2 saturations was observed for both large and small tumors of all lines. Saturations up to about 90% were also generally present, however, even in very large tumors. Variations in vascular configuration were predominantly tumor-line dependent rather than due to inherent characteristics of the host vasculature, and widely disparate HbO2 distributions were found for alternate lines implanted in identical host mice. Although peripheral saturations remained fairly constant with tumor growth, HbO2 values were markedly lower for vessels nearer the tumor center and further decreased with increasing tumor volume. HbO2 saturations did not change substantially with increasing vascular density (except for KHT tumors), although density did decrease with increasing distance from tumor surface. Combined effects of vessel diameter, tumor volume, and vessel location on HbO2 saturations were complex and varied markedly with both tumor line and vessel class. For specific classes, HbO2 distributions correlated closely with radiobiological hypoxic fractions, i.e., for tumor lines in which hypoxic fraction increased substantially with tumor volume, corresponding HbO2 values decreased, while for lines in which hypoxic fraction remained constant, HbO2 values also were unchanged. Although these trends may also be a function of differing oxygen consumption rates between tumor lines, functional alterations in the rapidly expanding tumor vasculature undoubtedly play a primary role in explaining spatial oxygenation heterogeneities.
Animals↗
31P nuclear magnetic resonance spectroscopy studies of tumor energy metabolism and its relationship to intracapillary oxyhemoglobin saturation status and tumor hypoxia.
Relationships between tumor bioenergetic status on the one hand and intracapillary oxyhemoglobin (HbO2) saturation status and fraction of radiobiologically hypoxic cells on the other were studied using two murine sarcoma lines (KHT, RIF-1) and two human ovarian carcinoma xenograft lines (MLS, OWI). Tumor energy metabolism was studied in vivo by 31P nuclear magnetic resonance (NMR) spectroscopy and the resonance area ratio (PCr + NTP beta)/Pi was used as parameter for bioenergetic status. Intracapillary HbO2 saturation status reflects the oxygen supply conditions in tumors and was measured in vitro using a cryospectrophotometric method. The KHT, RIF-1, and MLS lines showed decreasing bioenergetic status, i.e., decreasing PCr and NTP beta resonances and an increasing Pi resonance, with increasing tumor volume, whereas the OWI line showed no changes in these resonances during tumor growth. The volume-dependence of the HbO2 saturation status differed similarly among the tumor lines; HbO2 saturation status decreased with increasing tumor volume for the KHT, RIF-1, and MLS lines and was independent of tumor volume for the OWI line. Moreover, linear correlations were found between bioenergetic status and HbO2 saturation status for individual tumors of the KHT, RIF-1, and MLS lines. These observations together indicated a direct relationship between 31P-NMR spectral parameters and tumor oxygen supply conditions. However, this relationship was not identical for the different tumor lines, suggesting that it was influenced by intrinsic properties of the tumor cells such as rate of respiration and ability to survive under hypoxia. Similarly, there was no correlation between bioenergetic status and fraction of radiobiologically hypoxic cells across the four tumor lines. This indicates that 31P-NMR spectroscopy data have to be supplemented with other data, e.g., rate of oxygen consumption, cell survival time under hypoxic stress, and/or fraction of metabolically active, nonclonogenic hypoxic cells, to be useful in quantitative determination of tumor hypoxia and hence prediction of tumor radioresistance caused by hypoxia.
Animals↗
Glucose diffusivity in multicellular tumor spheroids.
In order to understand the role of glucose limitations in controlling multicellular tumor spheroid growth, knowledge of the glucose diffusion coefficient is essential. The effective diffusivity of glucose in spheroids of rodent and human tumor cell lines has been determined by measuring the efflux of tritium labeled L-glucose from spheroids with time. When the rapid and irreversible binding of L-glucose in spheroids is properly taken into account, measurements of the efflux of this diffusion tracer from spheroids into label-free medium can be correlated to the diffusion equation in order to obtain the effective glucose diffusivity in spheroids. Such measurements have been made in EMT6/Ro mouse mammary tumor spheroids as well as in spheroids derived from human colon carcinoma cells (HT29, CO112, and WiDr) and from human squamous carcinoma cells (CaSki and A431). EMT6/Ro spheroids have a glucose diffusivity of 1.1 x 10(-6) cm2/s, while glucose diffusion coefficients in the human cell spheroids studied vary from 5.5 x 10(-7) cm2/s to 2.3 x 10(-7) cm2/s. These values are low enough to suggest that significant gradients in glucose concentration may exist in spheroids and tumors. It is thus believed that these glucose diffusivities, as well as their variation with cell line, may have important implications for the role played by glucose in the growth and cellular heterogeneity of spheroids and tumors.
Algorithms↗
Cell and environment interactions in tumor microregions: the multicell spheroid model.
Abnormal vascularization of malignant tumors is associated with the development of microregions of heterogeneous cells and environments. Experimental models such as multicell spheroids and a variety of new techniques are being used to determine the characteristics of these microregions and to study the interactions of the cells and microenvironments. The special cellular microecology of tumors influences responsiveness to therapeutic agents and has implications for future directions in cancer research.
Antineoplastic Agents↗
31P NMR spectroscopy measurements of human ovarian carcinoma xenografts: relationship to tumour volume, growth rate, necrotic fraction and differentiation status.
31P NMR spectroscopy was used to study lipid and energy metabolism as well as tumour pH in three human ovarian carcinoma xenograft lines with widely differing growth rate, necrotic fraction and differentiation status. Two of the lines showed decreasing PCr (phosphocreatine) and NTP beta (nucleoside triphosphates beta) resonances and an increasing Pi (inorganic phosphate) resonance with increasing tumour volume range 100-4000 mm (3). This decrease in bioenergetic status was accompanied by a decrease in tumour pH from 7.15 to about 6.95. The volume-dependence of these spectral parameters probably reflected increased nutritional deprivation and development of hypoxia and necrosis during tumour growth. The phosphomonoesters (PME) and phosphodiesters (PDE) resonances did not change significantly with tumour volume. The third xenograft line did not show changes in the intensity of any of the resonances during tumour growth, in agreement with the observation that necrotic fraction and tumour pH (about 7.0) remained constant over the entire volume range. The spectral parameters differed significantly among the xenograft lines at given tumour volumes, but no correlations with volume-doubling time, necrotic fraction or differentiation status were found. The xenograft lines showed less extensive volume-dependence of the spectral parameters than did the KHT and RIF-1 murine tumour lines under identical experimental conditions.
Animals↗
Radiation sensitivity of human ovarian carcinoma cell lines in vitro: effects of growth factors and hormones, basement membrane, and intercellular contact.
The radiation sensitivity of six established human ovarian carcinoma cell lines was determined in vitro under five different experimental conditions. Cells from exponentially growing monolayer cultures were assayed under three different post-irradiation culture conditions, i.e., conventional conditions on a plastic surface, in the presence of growth factors and hormones, and on a basement membrane. Multicellular aggregates were dissociated either immediately before or immediately after irradiation and assayed under conventional conditions. The radiation sensitivity differed considerably among the cell lines; the initial slope alpha ranged from 0.05 +/- 0.03 Gy-1 to 0.36 +/- 0.07 Gy-1 and the surviving fraction at 2.0 Gy from 0.32 +/- 0.06 to 0.78 +/- 0.06 under conventional conditions. There was no significant effect of the growth factors and hormones and of the basement membrane on the survival curves for any of the cell lines. Only one of the lines showed a significant intercellular contact effect. The presence of this effect required that the cells were grown as aggregates, but was independent of whether the irradiation was performed on dissociated or intact aggregates. The present work with established cell lines indicates that the outcome of an in vitro predictive assay for clinical radioresponsiveness of ovarian carcinomas would probably not vary significantly among the five experimental conditions studied here. However, further studies using cells isolated directly from human ovarian carcinoma surgical specimens are warranted.
Basement Membrane↗
Hypoxic toxicity of misonidazole in a glucose-6-phosphate dehydrogenase deficient mutant Chinese hamster ovary cell line.
The metabolic activation of misonidazole (MISO) and its effects on the hexose monophosphate pathway (HMP) and on cell viability were studied in hypoxic mutant Chinese hamster ovary (CHO) cells deficient in glucose-6-phosphate dehydrogenase and their parent wildtype cells. The metabolic activation of MISO was similar in both cell lines as indicated by the binding of 14C-MISO to the acid-insoluble fraction of these cells; it was decreased by the absence of glucose. In the wildtype CHO cells, MISO caused a significant stimulation of the activity of the HMP while in the mutant CHO cells no HMP activity was measurable, even in the presence of MISO. In both cell lines clonogenicity began to decline after 2 hr and trypan blue exclusion after 4 hr of hypoxic incubation. The effect of MISO on both parameters of cell viability was somewhat more pronounced in the wildtype CHO cells. This difference became especially significant at the longer incubation times. The results indicate that reducing equivalents for the metabolic activation of MISO are provided not only by the HMP but that pathways other than the HMP, such as glycolysis or pathways starting from mitochondrial tricarboxylates, are of similar or even greater importance in this respect.
Animals↗
Growth phase related variation in the radiation sensitivity of human colon adenocarcinoma cells.
Changes in radiation sensitivity with length of time in culture are described for an early passage human colon adenocarcinoma cell line (WiDr). The cells were most radioresistant at the end of lag-phase, 2 days after subculture (Do = 1.7 Gy; n = 10). Radiation sensitivity then increased with time reaching a maximum during plateau-phase, between days 8 and 10 (Do = 1.0 Gy; n = 13). The oxygen enhancement ratio remained constant across the different growth phases of the culture. Cell volume decreased with time in culture as did the proportion of S- and G2M-phase cells. Flow cytometric analyses revealed an increase in the proportion of G1 type cells with a plateau between days 8 and 12 of around 75%. The cell age response measured from synchronized cells following 8 Gy showed that WiDr cells were most radiation resistant in mid S-phase with maximal sensitivity during G1. These cells did not show repair of potentially-lethal radiation damage but were efficient in the repair of sub-lethal damage. The ability to repair sub-lethal damage did not change with culture age.
Adenocarcinoma↗
Mathematical modelling of oxygen supply and oxygenation in tumor tissues: prognostic, therapeutic, and experimental implications.
Radiation response of some tumors is dependent on the oxygenation of the tumor tissue. To improve tissue oxygenation, attempts to increase a reduced hemoglobin concentration or to shift the dissociation curve of hemoglobin have been made. The aim of this paper is to estimate the influence of such measures on the volume of radiobiologically hypoxic tissue by means of a mathematical model. In addition, the influence of blood velocity and metabolic status of tumor tissues on tissue oxygenation is evaluated. The calculations show a strong influence on hypoxic tissue volume over a modest range of variation of physiological parameters, especially when interactions of several parameters may occur.
Hematocrit↗
Intracapillary HbO2 saturations in murine tumours and human tumour xenografts measured by cryospectrophotometry: relationship to tumour volume, tumour pH and fraction of radiobiologically hypoxic cells.
Frequency distributions for intracapillary HbO2 saturation were determined for two murine tumour lines (KHT, RIF-1) and two human ovarian carcinoma xenograft lines (MLS, OWI) using a cryospectrophotometric method. The aim was to search for possible relationships between HbO2 saturation status and tumour volume, tumour pH and fraction of radiobiologically hypoxic cells. Tumour pH was measured by 31P NMR spectroscopy. Hypoxic fractions were determined from cell survival curves for tumours irradiated in vivo and assayed in vitro. Tumours in the volume range 100-4000 mm3 were studied and the majority of the vessels were found to have HbO2 saturations below 10%. The volume-dependence of the HbO2 frequency distributions differed significantly among the four tumour lines; HbO2 saturation status decreased with increasing tumour volume for the KHT, RIF-1 and MLS lines and was independent of tumour volume for the OWI line. The data indicated that the rate of decrease in HbO2 saturation status during tumour growth was related to the rate of development of necrosis. The volume-dependence of tumour pH was very similar to that of the HbO2 saturation status for all tumour lines. Significant correlations were therefore found between HbO2 saturation status and tumour pH, both within tumour lines and across the four tumour lines, reflecting that the volume-dependence of both parameters probably was a compulsory consequence of reduced oxygen supply conditions during tumour growth. Hypoxic fraction increased during tumour growth for the KHT, RIF-1 and MLS lines and was volume-independent for the OWI line, suggesting a relationship between HbO2 saturation status and hypoxic fraction within tumour lines. However, there was no correlation between these two parameters across the four tumour lines, indicating that the hypoxic fraction of a tumour is not determined only by the oxygen supply conditions; other parameters may also be important, e.g. oxygen diffusivity, rate of oxygen consumption and cell survival time under hypoxic stress.
Animals↗
31P NMR spectroscopy in vivo of two murine tumor lines with widely different fractions of radiobiologically hypoxic cells.
Energy and lipid metabolism as well as tumor pH in two murine tumor lines, the KHT and RIF-1 sarcomas, were studied using 31P NMR spectroscopy. Possible relationships between spectral parameters on the one hand and volume fraction of necrosis and fraction of radiobiologically hypoxic cells on the other were investigated. For both tumor lines the PCr and NTP beta resonances decreased and the Pi resonance increased significantly with increasing tumor volume in the volume range 100-4000 mm3. This decrease in bioenergetic status was accompanied by a decrease in tumor pH from about 7.2 to about 6.8. The NTP beta resonance and the tumor pH tended to be somewhat higher and the Pi resonance somewhat lower for the KHT than for the RIF-1 tumors. Linear relationships were found between tumor pH and Pi or (PCr + NTP beta)/Pi for both tumor lines (P much less than 0.05). The PME resonance increased slightly and the PDE resonance decreased slightly during tumor growth and were not significantly different for the KHT and the RIF-1 tumors. The volume fraction of necrosis was about 5 per cent in both lines at a tumor volume of 100 mm3 and increased to about 30 per cent (KHT) and 50 per cent (RIF-1) at a tumor volume of 4000 mm3. The fraction of radiobiologically hypoxic cells was found to increase from 12 to 23 per cent for the KHT line and from 0.9 to 1.7 per cent for the RIF-1 line when tumor volume was increased from about 200 to about 2000 mm3. The volume-dependence of the 31P NMR spectral parameters indicated increased nutritional deprivation and development of hypoxia and necrosis during tumor growth, and was thus qualitatively in good agreement with the changes observed in necrotic and hypoxic fraction. However, quantitative relationships between any spectral parameter and necrotic or hypoxic fraction across tumor lines were not found, implying that other physiological parameters and/or cellular characteristics may contribute significantly to a 31P NMR tumor spectrum. Consequently, 31P NMR spectra of untreated tumors have to be supplemented with other tumor data, e.g. rate of oxygen consumption, cell survival time under hypoxic stress and/or fraction of metabolically active, non-clonogenic hypoxic cells, to be useful in quantitative determination of tumor hypoxia and hence prediction of tumor radioresistance caused by hypoxia.
Animals↗
Nutrient modification of proliferation and radiation response in EMT6/Ro spheroids.
The effects of external nutrients on the growth and radiation response of EMT6/Ro spheroids were studied by maintaining spheroids in media with different concentrations of glucose, amino acids, and vitamins. Compared to spheroids grown in normal glucose concentration (5.5 mM), spheroids grown in higher glucose media (24.8 mM), demonstrated no difference in initial volume doubling time, clonogenicity, number of proliferating cells, or cell cycle distributions. However, histology sections revealed that, spheroids grown in higher glucose concentration had a thicker viable rim than spheroids grown in normal glucose media. Two-step acridine orange staining and dual parameter flow cytometric analysis, in addition to continuous [3H]-thymidine labeling techniques, showed that spheroids grown in higher glucose had 2 to 3 times the fraction of quiescent cells, when compared to normal glucose spheroids. When irradiated in ice to reoxygenate, the Do's were similar in the normal and the higher glucose spheroids, but the Dq's were reduced in the higher glucose spheroids in the presence of increased amino acids and vitamins. When irradiated in air at 37 degrees C, spheroids grown in the higher glucose media were more sensitive (decreased Do), and had a smaller hypoxic fraction than when grown in normal glucose media. For spheroids grown in the same glucose concentrations but increased concentrations of amino acids and vitamins, there was generally an increased Do under all irradiation conditions. Some of these differences in radiation sensitivity could be correlated to differences in cellular glutathione levels of these spheroid cells.
Animals↗
Characterization of radiation sensitivity of human squamous carcinoma A431 cells.
Studies have been performed to investigate the radiosensitivity of human squamous carcinoma cells. A431 cells were grown in vitro as exponential and fed-plateau monolayer cultures or as multicellular spheroids. Radiobiological studies of various cultures showed that fed-plateau phase cells were more sensitive (D0 = 1.3 Gy) than exponentially growing cells (D0 = 1.5 Gy). After a single dose of 12 Gy or two doses of 6 Gy irradiation, A431 cultures exhibited a large capacity for potentially lethal damage (PLD) repair (PLD repair factor = 17), but a relatively small sublethal damage (SLD) repair. In order to measure the radiation sensitivity of proliferating (P) and quiescent (Q) cells, enriched populations of P- and Q-cells were isolated from A431 spheroids. Flow cytometric analysis with acridine orange (AO) staining demonstrated that there was a shift of the RNA histograms in fed-plateau and spheroid cultures towards lower values, suggesting the presence of a subpopulation of Q-cells. Centrifugal elutriation was used to isolate the Q-cells from dissociated spheroid cells. Coulter cell volume distributions and flow cytometric analysis showed that Q-cells had a small cell volume (approximately 1380 microns3), low RNA content and a G1-like DNA content. Continuous labelling experiments with tritiated thymidine confirmed the non-proliferating nature of the Q-cells. Irradiation of the Q-cells after isolation from spheroids with between 0 to 10 Gy showed that they were more radiosensitive (decreased D0) than the P-cells isolated from these spheroids. The latter were, however, similar in radiosensitivity to exponential G1 cells.
Carcinoma, Squamous Cell↗
Comparative pO2 measurements in cell spheroids cultured with different techniques.
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Animals↗
Dependence of misonidazole binding on factors associated with hypoxic metabolism.
The binding of misonidazole (MISO) to macromolecules in hypoxic cells is believed to require metabolic reduction. Several factors in the cells' environment, such as pH, glucose, lactate and MISO concentration could affect the capacity of metabolic reduction. Modulation of the binding of MISO by these factors was studied by exposing exponential EMT6/Ro cells to MISO under extremely hypoxic conditions. No binding was observed under aerobic conditions. There was no difference in the binding of 0.02 mM MISO at varying concentrations of glucose from 0.015 mM to 5 mM. Thus, for diagnostic purposes with concentrations of MISO lower than 0.02 mM, little effect of glucose concentration is expected. However, with 5 mM MISO, the binding of MISO increased with increasing glucose concentration (3-fold increase after 2 hours incubation in 5 mM glucose relative to 0.015 mM glucose). At intermediate MISO concentrations (0.1 mM to 5 mM); the higher the MISO concentration, the greater was the increase in binding due to 5 mM glucose. There was no detectable effect of lactate (0, 3 and 10 mM) at pH 7.2 on the binding of MISO either in 0.015 mM or 5 mM glucose. However, a decrease of pH (from 7.2 to 6.5) decreased the binding of MISO in 5 mM glucose but not in 0.015 mM glucose. These data indicated that the binding of MISO is a multi-step process, which involves the concentrations of both glucose (probably via reducing equivalents) and MISO.
Animals↗
Evaluation of CCNU and misonidazole in the treatment of advanced colorectal cancer.
Eighteen adult colorectal cancer patients, previously untreated with systemic chemotherapy, were given CCNU and MISO. One patient had an excellent partial response of pulmonary metastases, but the overall response rate was only 6%. Gastrointestinal toxicity was modest, hematologic toxicity was similar to what would have been predicted for CCNU alone, and there was no neurotoxicity detected. This Phase II study demonstrates that these two agents can be administered safely, but have no advantage over CCNU alone.
Adult↗