Multicell Tumor Spheroids Research Articles

Coupled cellular trafficking and diffusional limitations in delivery of immunotoxins to multicell tumor spheroids

Coupled cellular trafficking and diffusional limitations in delivery of immunotoxins to multicell tumor spheroids

Singlet Oxygen‐Versus Nonsinglet Oxygen‐Mediated Mechanisms of Sensitizer Photobleaching and Their Effects on Photodynamic Dosimetry

We report the effects of singlet oxygen (1O2) and non-1O2-mediated sensitizer photobleaching on oxygen consumption and dosimetry during photodynamic therapy (PDT) of sensitized multicell tumor spheroids. We develop a theoretical model for the description of non-1O2-mediated photobleaching resulting from irreversible reactions of the excited singlet or triplet sensitizer populations with cell substrate. We show that the fluence-dependent simple exponential decay expression of sensitizer degradation is not consistent with these mechanisms and, therefore, with any reasonable mechanism that we consider, because we have shown previously that 1O2-mediated photobleaching cannot be described by a simple exponential with a constant photobleaching coefficient (I. Georgakoudi et al., Photochem. Photobiol. 65, 135-144, 1997). Analysis of oxygen microelectrode measurements performed at the edge of Nile blue selenium (EtNBSe)- and protoporphyrin IX (PpIX)-sensitized spheroids during PDT demonstrates that the former drug photobleaches via a non-1O2-mediated mechanism, while the latter is degraded via a 1O2-mediated mechanism. Comparisons of the cytotoxic effects of EtNBSe with those of Photofrin (a drug that is degraded via a 1O2-mediated mechanism) indicate that the lower threshold 1O2 dose and the higher extinction coefficient and 1O2 yield for EtNBSe do not necessarily result in improved photodynamic effects, thus emphasizing the importance of the sensitizer photobleaching mechanism for dosimetry.

Cytotoxic Effects of High Energy Shock Waves on Cancer Cells Linked to Metallic Beads Vehicled by Monoclonal Antibodies

Previous studies have reported that high energy shock waves (HESW), generated by an electrohydraulic lithotriptor, may have some utility as a cancer treatment modality. Furthermore, it has been described that shock waves propagating in a fluid, show demolitive effects at the level of the interface of a solid fragment immersed in the fluid. In this study, we demonstrate that it is possible to enhance the antineoplastic effects of HESW if treated cells or tissues are linked to monoclonal antibodies (MoAbs) conjugated with metallic beads (MB) (about 1 mu of diameter) and specific for a cancer cell surface determinant. A leukemic cell line was used to study the effects of HESW on cells linked to MB. A fresh human breast cancer specimen was used to perform the assay on tumor tissue. MB linked treated cell viability, growth curve, cloning efficiency and Bromodeoxyuridine incorporation were reduced in comparison to cells treated with HESW alone. Our data suggest that the presence of solid fragments vehicled by MoAbs on a cancer cell surface is able to synergize with the limited antineoplastic effects of HESW.

Conceptual frameworks for mathematical modeling of tumor growth dynamics

The paper discusses the applicability of some empirical and functional mathematical models of tumor growth to multicell tumor spheroids which are a three-dimensional experimental paradigm of prevascular tumors. A new mathematical model based on receptor-mediated regulation of growth is developed and tested and two additional functional models (autostimulation, competing populations) are analyzed. It is demonstrated that these models, defined by two autonomous differential equations, can describe spontaneous tumor regression. The concept of tumor magnification factor, a quantifier of positive feedback mechanisms, is elaborated.

Somatostatin analogue octreotide modulates metabolism and effects of 5-fluorouracil and 5-fluorouridine in human colon cancer spheroids

To examine if preferential retention of somatostatin analogues observed in some tumors might be used for modulation of effects of cancer drugs by co-treatment with long acting somatostatin analogues, the effects of somatostatin analogue octreotide on the kinetics of 5-fluorouracil (FUra) and 5-fluorouridine (FUrd) metabolism were studied by 19F NMR spectroscopy in multicell tumor spheroids comprised of human colon HT-29 adenocarcinoma cells. Octreotide stimulated the rat of formation of fluorouridinephosphates in FUra-treated cells, but inhibited this rate in FUrd-treated cells. Other elements of fluoropyrimidine metabolism were also altered by co-incubation with octreotide. A flow cytometric analysis indicated that FUra and FUrd arrested cells in the S phase, but co-treatment with octreotide almost eliminated the S-phase cells and induced the appearance of DNA fragments. These observations raise the possibility that somatostatin analogues can be used for specific modulation of fluoropyrimidine effects in tumors bearing somatostatin receptors.

Comparisons of sectioned micro-TLD dose measurements with predicted dose from 131I-labeled antibody.

The dose distribution from radioimmunotherapy is very heterogeneous because of variability in antigen expression, antibody penetration, and tumor architecture. Many models of dose distribution have been constructed but it has been very difficult to confirm these predictions with actual measured doses. The purpose of this study was to determine what degree of resolution could be obtained using mini-thermoluminescent dosimeter(s) (TLD) in a micrometastasis model. TLDs were inserted into 1-mm-diam multicell tumor spheroids that had been treated with 131I-labeled antibody. The spheroids were then sectioned at 30-microns intervals and the TLD sections (measuring 0.14 x 0.1 x 0.03 mm) were removed and read. Calibration of the TLDs was done with whole TLDs using external beam radiation and an 131I-containing gel, and with TLD sections using external beam radiation. Predicted doses were determined by measuring the activity in individual spheroids and the time the TLDs were in the spheroids and incorporating these numbers into a model that assumed either surface binding of 131I or some degree of penetration. There was a correlation between the measured TLD dose and the predicted absorbed dose when comparing the low- to high-dose regions. However, there was considerable variation within any particular dose range, probably due to heterogeneity in TLD grain size.(ABSTRACT TRUNCATED AT 250 WORDS)

Activity of the human metallothionein promoter (hMT-II A) in cell populations isolated from varying depths in multicell spheroids following exposure to cadmium

In order to evaluate whether tumor microenvironment might influence the response of the metallothionein promoter to heavy-metal exposure, we transfected HT-29 colon carcinoma cells with the vector phMTII A-CAT-neo, containing a fusion gene consisting of 426 bp of the human metallothionein-IIa (hMT-II A) promoter immediately upstream of the bacterial chloramphenicol acetyl transferase (CAT) gene. We grew one of the stable transfectants (clone 20) as three-dimensional multicell tumor spheroids, exposed them to CdCl 2 and measured CAT expression in cells isolated from various depths into the spheroids. Cellular populations were isolated by flow cytometry on the basis of Hoescht 33342 fluorescence intensity, taking advantage of the dye's diffusion gradient to isolate cells from inner (dim) and outer (bright) regions of stained spheroids. When intact spheroids were incubated for 18 h in the presence of 5 μM CdCl 2, CAT activity was induced in all cell fractions isolated from the spheroids, but induction was 10-fold greater in cells in the outermost fraction (fraction 10) than inner fraction (fraction 2). When spheroids were dissociated, sorted into individual fractions and then incubated with cadmium, CAT expression was maximized in all fractions. Exposure of intact spheroids to 30 μM CdCl 2 resulted in increased CAT induction in cells isolated from the internal fractions of the spheroids. The data suggest that limited diffusion of cadmium through cells organized in a tissue-like arrangement may account for the lower levels of hMT-II A promoter activity observed in cells collected from increasing depths into the spheroids.

Histopathology of shock wave treated tumor cell suspensions and multicell tumor spheroids

L1210 mouse leukemia cell suspensions exposed to 500 shock waves (SW) in an experimental lithotripter (XLI, Dornier) revealed severe cellular damage. Apart from cell fragments and cellular debris, cells exhibited alterations of shape, vacuolisation of the cytoplasm, perinuclear cisternae, swelling of mitochondria or rupture of the mitochondrial fine structure, and permeabilization of the cell membrane. Treatment of multicell tumor spheroids of both HeLa and EMT6/Ro cells in suspension with 500 SW resulted either in loss of peripheral cells and serious cellular damage in the outer regions or in a fragmentation of the spheroids. Many of the geometrically intact cells exhibited the same histopathological alterations as the suspended L1210 cells. Immobilization of the spheroids in agar or gelatine, however, prevented spheroids from being agitated and accelerated during SW-exposure. After treatment with 500 SW, spheroids immobilized in gelatine were not different from control cultures, as investigated with light- and electronmicroscopy. From our results we conclude that spheroids in suspension are subject to cavitation and liquid jet formation, causing not only acceleration and shearing forces but also collisions which account for the observed cell damage.

Recovery from Potentially Lethal Damage and Recruitment Time of Noncycling Clonogenic Cells in 9L Confluent Monolayers and Spheroids

Cells that have been grown as multicell tumor spheroids exhibit radioresistance compared to the same cells grown in monolayers. Comparison of potentially lethal damage (PLD) repair and its kinetics was made between 9L cells grown as spheroids and confluent monolayers. Survival curves of cells plated immediately after irradiation showed the typical radioresistance associated with spheroid culture compared to plateau-phase monolayers. The dose-modification factor for spheroid cell survival is 1.44. Postirradiation incubations in normal phosphate-buffered saline (PBS), conditioned media, or 0.5 M NaCl in PBS reduced the differences in radiosensitivity between the two culture conditions. Postirradiation treatment in PBS or conditioned medium promoted repair of potentially lethal damage, and 0.5 M NaCl prevented the removal of PLD and allowed the fixation of damage resulting in lower survival. Survival of spheroid and monolayer cells after hypertonic NaCl treatment was identical. NaCl treatment reduced Do more than it did the shoulder (Dq) of the survival curve. PLD repair kinetics measured after postirradiation incubation in PBS followed by hypertonic NaCl treatment was the same for spheroids and for plateau-phase monolayers. The kinetics of PLD repair indicates a biphasic phenomenon. There is an initial fast component with a repair half-time of 7.9 min and a slow component with a repair half-time of 56.6 min. Most of the damage (59%) is repaired slowly. Since the repair capacity and kinetics are the same for spheroids and monolayers, the radioresistance of spheroids cannot be explained on this basis. Evidence indicates that the time to return from a Go (noncycling G1 cells) state to a proliferative state (recruitment) for cells from confluent monolayers and from spheroids after dissociation by protease treatment may be the most important determinant of the degree of PLD repair that occurs. Growth curves and flow cytometry cell cycle analysis indicate that spheroid cells have a lag period for reentry into a proliferative state. Since plating efficiency remains high and unchanging during this period, one cannot account for the delay on the basis of the existence of a large fraction of Go cells which are not potentially clonogenic. The cell cycle progression begins in 6-8 h for monolayer cells and in 14-15 h for spheroids. It is hypothesized that the slower reentry of spheroid cells into a cycling phase allows more time for repair than for the rapidly proliferating monolayer cells.

Evidence for a major role of glucose in controlling development of necrosis in EMT6/Ro multicell tumor spheroids.

Multicell tumor spheroids show many features of tumors in vivo such as saturation of growth, induction of quiescent cells (Freyer and Sutherland, 1980; Bauer et al., 1981) and development of necrosis (Sutherland and Durand, 1976; Franko and Sutherland, 1979b; Carlsson et al., 1979). These effects may be attributed to limited penetration of metabolites into the spheroid due to cellular consumption and due to restricted diffusion as the spheroid increases in size (Mueller-Klieser and Sutherland, 1981b). Many investigations of cells in vitro and in experimental tumors in vivo (Vaupel, 1980) have indicated that oxygen plays a decisive role in the control of the metabolism and the viability of cancer cells. It has also been demonstrated in spheroids that the thickness of the viable rim surrounding the necrotic center depends on the oxygen concentration in the culture medium (Franko and Sutherland, 1979a). Thus, oxygen can be a critical determinant for the development of necrosis in multicell tumor spheroids. Recent measurements, however, of pO2 values in spheroids using oxygen sensitive microelectrodes (Mueller-Klieser and Sutherland, 1981b) have demonstrated that necrosis may develop at different levels of oxygen tensions as the spheroids increase in size. This indicates that additional factors other than oxygen may also be involved in the control of cell death in spheroids. The present study was undertaken to investigate the influence of oxygen and glucose concentrations in the culture medium on the thickness of the viable rim and on the oxygen tensions in EMT6/Ro spheroids.KeywordsOxygen TensionTumor SpheroidCentral PlateauSpinner FlaskControlling DevelopmentThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Determination of diffusion constants for metabolites in multicell tumor spheroids.

Multicell tumor spheroids (MTS) exhibit many of the characteristics of tumors, including central necrosis, heterogeneities in proliferative status and clonogenic capacity, and subpopulations of radiation and chemotherapeutic resistant cells (Freyer and Sutherland, 1980; Sutherland and Durand, 1976). In tumors, control of most of these parameters appears to be due to low molecular eight nutrients such as oxygen and glucose. Tannock (1976) suggested that loss of proliferative activity was correlated with the oxygen diffusion distance. Low values of oxygen partial pressure in tumors are common (Thews and Vaupel, 1974), as are low pH values (Vaupel et al., 1981). Tumor extracellular fluid has been shown to contain low levels of glucose (Guillino, 1975). Most tumors are very heterogeneous with respect to the local microenvironment (Mueller-Klieser et al., 1980) due to the random structure and chaotic functioning of the vascular system. Thus, it has been proven difficult to establish precisely which nutrient(s) are critical for cellular proliferation and viability in vivo.

The role of glucose in the growth of 9L multicell tumor spheroids.

Monolayer-cultured 9L rat brain tumor cells grew optimally in culture media containing glucose concentrations greater than 0.07 mg/ml, but growth ceased at concentrations lower than this value. Assuming that this glucose concentration defines the proliferating fraction of cells in 9L multicell tumor spheroids, it is possible to simulate spheroid growth based on the intraspheroid glucose distribution derived by the author earlier. An excellent fit to experimental growth data was obtained by assigning the proliferating fraction a growth rate constant equal to 40% of that of monolayer-cultured cells. This definition is in accord with our observation that the colony-forming efficiency (CFE) of spheroid-derived cells is 40% of the CFE of cells maintained in monolayer. This suggests that the role of glucose in 9L cell growth is similar in spheroids and in monolayer culture.

The glucose distribution in 9L rat brain multicell tumor spheroids and its effect on cell necrosis.

The glucose consumption rate of 9L rat brain tumor cells was determined as a function of concentration. Glucose uptake followed Michaelis-Menten kinetics with a Km of 0.58 mM and a Vmax of 1.6 pg/cell-min. The glucose diffusion coefficient in spheroids of 9L tumor cells was determined to be 1.5 x 10(-6) cm2/s at 37 degrees C. Using these parametric values, the glucose distribution in 9L multicell spheroids was calculated and related to the viable and necrotic zones.

Influence of growth, measuring conditions, and cell types on oxygen tensions in multicell tumor spheroids determined by microelectrodes

Influence of growth, measuring conditions, and cell types on oxygen tensions in multicell tumor spheroids determined by microelectrodes

Density gradient centrifugation of cells separated from multicellular tumor spheroids.

Cells from a murine fibrosarcome (FSa) have been grown in vitro as multicell tumor spheroids (MTS). The growth rate of these MTS was determined. Following selected periods of growth, MTS were made into a single cell suspension and separated on linear density gradients of Renografin. While only 1 population of cells were separated from small spheriods (400 mum diameter), at least 3 subpopulations of tumor cells were separated and isolated from large spheroids (800 mum in diameter).