Biomedical subjects

R M Sutherland

Publications and source records attributed to R M Sutherland.

At least 109 records · Page 6Linked to original sources

PubMed1987

The role of glycolysis and hexose monophosphate pathway in the hypoxic toxicity of misonidazole.

The metabolic activation of misonidazole (MISO) and its effects on the hexose monophosphate pathway (HMP) and clonogenicity were studied in hypoxic EMT6/Ro, wildtype Chinese hamster ovary (CHO) and mutant CHO cells deficient in glucose-6-phosphate dehydrogenase. In all three cell lines metabolic activation of MISO, as indicated by the binding of 14C-MISO to the acid-insoluble fraction of these cells, was increased by the presence of glucose. In EMT6/Ro cells and 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. Loss of clonogenicity induced by MISO occurred markedly earlier in EMT6/Ro cells than in the CHO cells. In the latter cells, however, only a small difference was observed between the wildtype and mutant cell line. From these results it is concluded that not only the HMP but also glycolysis and other, glucose-independent, metabolic pathways are able to provide electrons for the reductive activation of MISO and hence contribute to the hypoxic toxicity of this compound.

Animals↗

PubMed1987

A human colon adenocarcinoma xenograft--radiation response, cellular composition, and tumor disaggregation.

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

Adenocarcinoma↗

PubMed1987

The radiation response of a human colon adenocarcinoma grown in monolayer, as spheroids, and in nude mice.

A human colon adenocarcinoma cell line, WiDr, has been grown in monolayer, as multicellular spheroids, and as xenografted tumors in immune-deprived mice. The growth and radiation responses of the cells under these different growth conditions were compared. The mean doubling time of monolayer cultures was 0.8 day and the initial volume doubling times of spheroids and xenografts averaged 1.2 and 6 days, respectively. The mean total viable cell plating efficiencies were 82, 63, and 7% for cells from monolayers, spheroids, and xenografted tumors, respectively. The radiation responses of single cell suspensions prepared from WiDr tumors (8-10 mm in diameter), exponentially growing monolayer cultures (5 days growth), and spheroids (1200 microns in diameter) irradiated in air at 4 degrees C were similar. Values for D0 were 1.5 Gy and for n between 3 and 5. Nitrogen curves were characterized by a D0 of 5 Gy and n between 3 and 6. Oxygen enhancement ratios were approximately 3.3. Both spheroids and tumors had radioresistant components to the 37 degrees C/air-breathing survival curves with estimated hypoxic fractions of 8 and 12%, respectively. The final portion of the survival curves for irradiations in nitrogen and under normal growth conditions were parallel for both tumors and spheroids. Thus WiDr spheroids appear to model accurately the radiation sensitivity of WiDr tumors.

Adenocarcinoma↗

PubMed1986

Importance of critical metabolites and cellular interactions in the biology of microregions of tumors.

Heterogeneity of cellular microenvironments and associated differences in phenotypic expression of cells in solid tumors occur as a consequence of deficiencies of vascularization. Intervascular microregions of tumors and micrometastases can be modeled by multicellular spheroids in vitro. These develop concentration gradients of critical metabolites such as oxygen, glucose, and probably also other important nutrients, hormones, and growth factors. Drug penetration may also be reduced. Concentrations of these factors decrease from the periphery towards the center, but gradients develop in the opposite direction for certain metabolic and necrotic products such as lactate and pH. Such gradients significantly modify the proliferative status of the cells, viability, clonogenicity, cell cycle distribution, antigen expression, and differentiation. Stress of oxygen and glucose deprivation induces synthesis of a specific set of proteins. Cellular interactions can also modulate phenotypic expression, including oxygen and glucose consumption rates and sensitivity to radiation and drugs. The mechanisms of these are unknown, but changes in cell-cell communication and DNA damage and repair have been demonstrated in spheroids. Microenvironmental and cellular changes may be transient. Thus, the characteristics of cells in tumor microregions may be quite different from the intrinsic cellular properties observed after growth of cells in standard culture systems. These changes may have significant effects on responses to therapeutic agents.

Adenocarcinoma↗

PubMed1986

Density distributions of human squamous carcinoma cells: influence of growth conditions, proliferative status and DNA content.

Human squamous carcinoma cells (A431, CaSki) were grown in vitro as multicellular spheroids or as exponential and plateau-phase monolayer cultures. Single-cell suspensions were obtained by disaggregating the spheroids and monolayer cultures with trypsin-EDTA. Their buoyant density distributions were then compared by centrifuging the suspensions for 30 min at about 800gave and 4 degrees C in linear, continuous Percoll gradients. We found differences in buoyant density related to the growth condition (i.e. cells grown as spheroids or plateau-phase cultures were more dense than those from exponential cultures) and to the cell-line (i.e. CaSki cells were more dense than the A431 cells). Cells isolated from the inner layers of the spheroids appeared to be less dense than those from the outer layers. In addition, quiescent cells separated from the A431 spheroids by centrifugal elutriation, a technique based on cell size, were more dispersed in buoyant density than the corresponding proliferating cells recovered from the same spheroids. Flow cytometric analysis with mithramycin was performed to measure the DNA content of the cells. We found that the CaSki cells contained relatively more DNA than the A431 cells whereas, for the same cell line, growth as monolayer cultures or as multicellular spheroids did not influence their relative DNA content. We conclude that the growth conditions, the proliferative status of the cells and possibly their relative DNA content may influence the density distribution of these cells.

Carcinoma, Squamous Cell↗

PubMed1986

Influence of growth phase, nutrition and hypoxia on heterogeneity of cellular buoyant densities in in vitro tumor model systems.

EMT6/Ro multicellular spheroids were sequentially dissociated into outer, middle and inner layers, and the buoyant densities of these subpopulations were compared to aerobic or exponential or plateau-phase monolayers by isopycnic density centrifugation using Percoll as the gradient medium. Spheroids were more heterogeneous in buoyant density distributions than monolayer cultures. The outer-layer cells of spheroids and fed plateau monolayers peaked at a similar density; the majority of middle-layer cells banded close to unfed plateau and exponential hypoxic monolayers, while most inner-layer cells banded where fed or unfed plateau hypoxic monolayer peaked in density. Our data suggest that metabolic heterogeneity due to growth phase, together with altered cellular microenvironment brought about by differences in oxygenation and external nutrient supplies between spheroids and monolayers, can be manifested in one biophysical parameter such as buoyant density.

Animals↗

PubMed1986

A radiobiological comparison of human tumor soft-agar clonogenic assays.

Radiation survival curves have been generated for 3 human tumor cell lines as a means of comparing and evaluating the validity of human tumor soft-agar clonogenic assays. The assays investigated were the Hamburger-Salmon, Courtenay-Mills, Courtenay-Mills plus additions, soft agar (no additions), and soft agar plus additions. The additions were formulated to supplement the media used in soft agar assays of primary ovarian and cervical carcinoma specimens. Supplementing the media with additions led to a 2- to 3-fold increase in PE of CaSki cells but had no effect on the PEs of ME180 and OWI cells. Radiation survival curves were similar in all assays for CaSki and OWI but differed for ME180 cells. For ME180 cells, the Courtenay-Mills and soft agar assays plus additions produced the most radioresistant curves (Do = 2.2 Gy); the cells were more responsive when assayed by the Hamburger-Salmon method (Do = 1.5 Gy), and the soft agar and Courtenay-Mills assays gave the most radiosensitive curves (Do = 1.2 Gy). These results demonstrate that the PE of human tumor cell lines may be increased with no effect on radiation survival; radiation survival may be altered without changes in PE and neither may be altered by applying modifications and supplements to existing clonogenic assays.

Adenocarcinoma↗

PubMed1986

Induction characteristics of oxygen regulated proteins.

Extreme hypoxia induces many changes in the biology of cells, including the enhanced synthesis of oxygen regulated proteins (ORPs). We investigated the conditions required for the induction of ORPs and by modifying culture conditions, eliminated variables other than oxygen concentration. Several exponentially growing rodent and human cell lines were examined before, during, and after various periods of extreme hypoxia. The following responses were analyzed: cell growth, clonogenic survival, glucose consumption, lactate production, media pH, total protein synthesis, and specific protein synthesis. EMT6/Ro cells did not increase in cell number or progress through the cell cycle after initiation of extreme hypoxia. Cell morphology and cell survival were nearly normal for up to 12 hr of hypoxia. During this period, media pH remained constant, with the concentrations of glucose and lactate being virtually indistinguishable from aerobic cultures or initial values. Associated with these conditions, a marked inhibition of total protein synthesis was observed for EMT6/Ro cells, such that the hypoxic protein synthesis rate was about 60% of the aerobic rate. However, enhanced synthesis of a set of proteins, designated as ORPs, was preferentially induced in less than 6 hr. The molecular weights of the five major ORPs are 260, 150, 100, 80 and 33 kD. Under these conditions, the primary inducing agent was a low concentration of oxygen. This set of ORPs was distinctly different from the set of heat induced (heat-shock) proteins, but included the major 100 kD and 80 kD glucose regulated proteins. Although the functions of ORPs are unknown, their induction under conditions that are known to modify the sensitivity of cancer cells to therapeutic agents suggests that the presence of ORPs should be further investigated to determine their possible value in diagnosis and predicting treatment response.

Animals↗

PubMed1986

Theoretical evaluation of expected changes in oxygenation of tumors associated with different hemoglobin levels.

A mathematical computer-based model was developed which analyzes the influence of hemoglobin concentration; shape of the oxygen dissociation curve; red cell velocity; capillary network geometry; and oxygen consumption on the oxygen partial pressure (PO2) distribution in tissue. In this study, the influence of acute hemoglobin concentration changes on the oxygen supply to tissue was analyzed. The specific question, whether moderate changes in the hemoglobin concentration of only 20% (e.g., from 15 g/dl to 12 g/dl i.e. baseline is 15 g/dl), as they may often occur clinically, are able to produce a significant change in the tissue oxygenation, was addressed. This is of importance for the estimation of the possible effects of blood transfusions prior to radiation therapy. The model predicts that even a moderate 20% increase in the hemoglobin concentration is able to decrease the anoxic tissue volume by 30%. The results indicate that hemoglobin levels should not be subnormal at the beginning of chemotherapy or radiotherapy. Transfusions or the application of artificial oxygen carriers could be helpful.

Hemoglobins↗

PubMed1986

Radiation response of proliferating and quiescent subpopulations isolated from multicellular spheroids.

Two subpopulations enriched in cells with a G1-like content of DNA were isolated from EMT6/Ro spheroids using centrifugal elutriation. The techniques of two-step acridine orange staining followed by flow cytometry, and continuous [3H]-thymidine labelling agreed qualitatively that one of these subpopulations predominantly consisted of proliferating G1 cells, while the other contained about four times more quiescent G0/G1 cells. These two subpopulations had similar median cell volumes and DNA contents, but the cell volume distributions were different. The clonogenicity was greater in the 'proliferating' subpopulation than the 'quiescent' subpopulation. When cell number seeded was corrected for viability, regrowth studies showed that there was a longer time (25 h) for the 'quiescent' subpopulation than the 'proliferating' subpopulation (10 h) before any increase in cell number was observed. In addition, relative to the 'proliferating' cells, the 'quiescent' cells were more sensitive when exposed to 137Cs gamma-ray radiation. The D0's were similar between the two subpopulations (D0 = 1.6 Gy and 1.8 Gy for the 'proliferating' G1 and 'quiescent' G0/G1 subpopulation, respectively), but the width of the shoulder of the radiation survival curve was reduced in the 'quiescent' subpopulation (Dq = 2.3 Gy vs. 5.3 Gy).

Animals↗

PubMed1986

Modulation of the hypoxic toxicity and binding of misonidazole by glucose.

The hypoxic toxicity and binding of misonidazole (MISO) requires metabolic reduction. The influence of glucose on the toxicity and binding of MISO was studied because glucose is a major substrate for the supply of NADPH through the hexose monophosphate pathway (HMP). Hypoxic EMT6/Ro cells (10(6) cells ml-1) were incubated with varying concentrations of glucose (0.015 mM to 5 mM). The initial rate of glucose transport was found to increase linearly with the extracellular glucose concentration up to 5 mM (0.038 nmol glucose 10(-6) cells sec-1). About 1.5 percent of the total glucose consumed went through the HMP for hypoxic cells in 5 mM glucose. The rate of HMP progressively decreased as the glucose concentration was lowered. When exposed to 5 mM MISO, the HMP was stimulated. This stimulation declined from 3.2 times in 5 mM glucose to barely detectable below 1 mM glucose. Both the hypoxic toxicity and binding of 5 mM MISO to the acid-insoluble fraction were decreased as the concentration of glucose was lowered. Below 0.5 mM glucose, no significant toxicity due to MISO was observed. There was an initial burden of 2.5 nmol MISO 10(-6) cells bound with little toxicity. After this initial burden, the terminal slope was 1.8 mol MISO bound 10(-6) cells (63 percent decrease in the surviving fraction). These results indicate that glucose concentrations lower than 5 mM can decrease the HMP rate and the toxicity and binding of MISO to hypoxic cells, and imply that calibration curves with normal and low glucose concentrations should be used to estimate the possible hypoxic fraction when MISO is used as a hypoxic probe in vivo.

Animals↗

PubMed1986

Influence of glucose and oxygen supply conditions on the oxygenation of multicellular spheroids.

The interrelationship among external O2 and glucose supply, oxygenation status, oxygen consumption rates and cellular viability in tumour microregions was studied using the multicellular spheroid model. For chronic exposure to various supply conditions multicellular EMT6/Ro spheroids were cultured in stirred media equilibrated either with 20% (v/v) or 5% (v/v) oxygen and containing four different glucose concentrations ranging from 0.8 mM to 16.5 mM. Spheroids were investigated using histology and O2-sensitive microelectrodes for measuring oxygen tension (PO2) values. A chronic decrease of the glucose concentration in the medium is associated with a substantial reduction in the thickness of the viable rim of cells and with a persistent increase in the cellular respiration rate. In general, both viable rim size and respiration are decreased through restriction of O2 supply during spheroid growth at a given external glucose concentration. The O2 consumption in spheroids appears to decrease with increasing spheroid size under most of the growth conditions investigated. These findings provide evidence for a large capacity of the spheroid cells to chronically adapt their metabolic rates to different supply situations. The experimental data and theoretical considerations indicate that necrosis may develop in the centre of these spheroids due to the lack of O2 and/or glucose under some of the growth conditions, but central necrosis can also occur despite sufficient O2 and glucose supply. Consequently, cellular metabolism and viability in tumour microregions may not be determined by the diffusion limitation of O2 or specific substrates alone, such as glucose, but may be influenced by a complex interaction of factors in the micromilieu the majority of which are still unknown.

Animals↗

PubMed1986

Regulation of growth saturation and development of necrosis in EMT6/Ro multicellular spheroids by the glucose and oxygen supply.

To investigate the effects of glucose and oxygen on spheroid growth, EMT6/Ro mouse mammary carcinoma cell spheroids were cultured in suspension in either 0.28 mM (20%) or 0.07 mM (5%) oxygen and 16.5, 5.5, 1.7, and 0.8 mM glucose. The spheroids initially grew at the same exponential rate in all culture conditions, with spheroid volume and cell number doubling times of 20-24 h. The growth rates slowed as the spheroids grew, and the maximum volume and cell number attained at growth saturation were proportional to the oxygen and glucose concentrations in the medium. There was a 500-fold difference in saturation sizes comparing spheroids cultured in the highest oxygen and glucose concentrations to those grown in the lowest. The thickness of the viable cell rims was also positively correlated with the oxygen and glucose concentrations in the medium. Comparison of the growth saturation and viable cell rim data showed an excellent correlation between the onset of central necrosis and the cessation of spheroid growth. A model is presented to explain the observed spheroid growth characteristics by proposing a competition between externally supplied growth and viability-promoting factors and internally generated inhibitory factors produced by the process of necrosis. This model has critical implications for the use of spheroids as models of cellular growth in tumors.

Animals↗

PubMed1986

Proliferative and clonogenic heterogeneity of cells from EMT6/Ro multicellular spheroids induced by the glucose and oxygen supply.

The culture of EMT6/Ro multicellular tumor spheroids in a 20-fold range of glucose concentrations and a 4-fold range of oxygen tensions had profound effects on the cell subpopulations which developed in the spheroids. As the spheroids increased in diameter the rate of accumulation of cells with a G1-phase DNA content was much greater for those spheroids cultured in the lower oxygen and glucose concentrations. Growth fractions of 600- to 800-micron-diameter spheroids, calculated from either DNA content or [3H]thymidine labeling data, showed a direct correlation with the glucose concentrations in the culture medium. The cells from spheroids cultured in low glucose concentrations also showed a more rapid loss in clonogenicity as a function of increasing spheroid diameter. Lowering the oxygen tension enhanced these effects of reduced glucose concentrations. Selective dissociation of 600- to 800-micron-diameter spheroids demonstrated that both the proliferation index and the clonogenic capacity of the spheroid cells decreased with increasing depth into the spheroid cell rim. The rates of decrease of both of these parameters as a function of depth into the spheroid were greater for spheroids cultured in the lower oxygen and glucose concentrations. These results indicate that both the glucose and oxygen supply are critical in the development of nonproliferating and nonclonogenic cell subpopulations in spheroids.

Animals↗

PubMed1986

Oxygenation and differentiation in multicellular spheroids of human colon carcinoma.

Oxygenation and development of necrosis were evaluated in multicellular spheroids of poorly differentiated (HT29) and moderately well-differentiated (Co112) human adenocarcinoma of the colon. Spheroids were grown in vitro under well-controlled oxygen and nutrient conditions in spinner flasks up to sizes of 2800-micron diameter after 5 wk of culture. Morphological studies showed that the Co112 spheroids contained pseudoglandular structures with lumen, very similar to the characteristics of the original tumor specimen from the patient and to the cells when grown as xenograft tumors in nude mice. Microelectrodes were used to measure the oxygen tension (PO2) profile within individual spheroids at different stages of growth. Histological sections through the centers of spheroids were measured to determine the thickness of the viable rim of cells surrounding spheroid necrotic centers in order to estimate the size of the severely hypoxic zone of cells by comparison with the PO2 profiles of the same spheroids. The data demonstrate significant differences between these two human colon tumor spheroid systems. Both spheroid types exhibited steep PO2 gradients at relatively small sizes of less than 600-micron diameter, but for any given size in this range, the more differentiated Co112 spheroids were more hypoxic. Although severe hypoxia (PO2, less than 10 mm of Hg) was present in both spheroid types at larger sizes, there was a significant difference in the central PO2 values which were between 5 and 10 mm of Hg in large Co112 spheroids but remained at or close to 0 mm of Hg in large HT29 poorly differentiated human colon tumor spheroids. The presence of pseudoglandular structures and lumen in the Co112 spheroids was associated with changes in the shape of PO2 profiles. Such profiles have not previously been seen in other poorly differentiated human or rodent tumor spheroids. Furthermore, the PO2 profiles of both of these human tumor spheroid types were often continuously curving with a very shallow gradient in the inner edge of the viable rim of cells surrounding the necrotic center. Regulation of oxygen consumption and/or diffusion in these inner regions of human spheroids could produce these continuously curving PO2 gradients.

Adenocarcinoma↗

PubMed1986

In vitro and in vivo tumor models for studies of distribution of radiolabelled monoclonal antibodies and fragments.

Colon carcinoma multicellular spheroids were incubated in vitro with radiolabelled MAbs. The more rapid penetration of fragments as compared to intact MAbs was clearly demonstrated. For the study of antibody localization in tumors in vivo, the model of nude mice with ligated kidneys was used. Although very artificial, this model allowed to demonstrate that, without urinary excretion, Fab fragments accumulated more rapidly into the tumor than intact MAbs and disappeared faster from the blood. This difference was less striking for F(ab')2 fragments. In the liver a decreased accumulation of both types of fragments as compared to intact MAbs was observed. Concerning radioimmunotherapy we think that Fab fragments are not useful because of their too short half-life in the circulation and in tumor and because they will probably be too toxic for the kidneys. Intact MAbs and F(ab')2 fragments have each their advantages. Intact MAbs show highest tumor accumulation in mice without ligated kidney, however, they remain mostly on the periphery of tumor nodules, as shown by autoradiography. F(ab')2 fragments have been found to penetrate deeper into the tumor and to accumulate less in the liver. It might be therefore an advantage to combine intact MAbs with F(ab')2 fragments, so that in the tumor two different regions could be attacked whereas in normal tissues toxicity could be distributed to different organs such as to the liver with intact MAbs and to the kidney with F(ab')2 fragments.

Animals↗

PubMed1985

Radiation oncology. Programs for the present and future.

Radiation oncology in 1984 continues to make major advances in the multidisciplinary clinical programs. This has been possible by virtue of the radiation oncologist, who is an active participant in these clinical programs. The changing role for the radiation oncologist has dictated a greater participation in the primary management of the patient's disease process and also participation in multidisciplinary research programs.

Breast Neoplasms↗

PubMed1985

Increased thermoresistance developed during growth of small multicellular spheroids.

Mammalian cells growing as multicell spheroids, an in vitro model of tumor microregions, have been shown previously to be more resistant than single cells from monolayer cultures to killing by ionizing radiation, hyperthermia, ultrasound, and chemotherapeutic drugs. Although the mechanisms by which cells in spheroids acquire these increased resistances are unknown, available evidence has indicated that intercellular contact mediates the process for ionizing radiation. This investigation was undertaken to evaluate the role of intercellular contact produced during growth of small spheroids on the sensitivity of EMT6/Ro mouse mammary tumor cells to moderate hyperthermia. Increased thermoresistance developed in small spheroids (approximately 70 micron diameter, 25 cells/spheroid), as measured by colony formation, after exposures to different temperatures in the range of 37 to 45 degrees C for periods less than or equal to 2 hr and at 42.5 degrees C for less than or equal to 8 hr. Experiments were performed to determine the relative contributions to this increased thermoresistance of 1) the extent of intercellular contact in spheroids of different cellular multiplicities, 2) differences in membrane damage influenced by trypsin heat treatment sequence, and 3) physiological changes associated with growth of cells as spheroids in suspension compared to monolayer culture. Treatment with trypsin prior to heating sensitized cells to killing by hyperthermia but did not account for the differential thermoresistance between cells from spheroids and monolayers. Spheroid multiplicity in the range of 1.16 to 76.2 cells/spheroid had no significant effect on cell survival after hyperthermia. However, cells grown in spinner suspension culture were more thermoresistant than cells from monolayer cultures and nearly as thermoresistant as cells in spheroids. From these data we conclude that the greater thermoresistance of EMT/Ro cells in spheroids is the result of cellular physiological changes associated with growth in suspension and is not mediated by intercellular contact.

Animals↗