The effect of 238Pu alpha-particles on the mouse fibroblast cell line C3H 10T1/2: characterization of source and RBE for cell survival

Considerable interest has been aroused in recent years by reports that the transforming and carcinogenic effectiveness of low doses of high LET radiations can be increased by reducing the dose rate, especially for transformation of 10T1/2 cells in vitro by fission-spectrum neutrons. We report on conditions which have been established for irradiation of 10T1/2 cells with high LET monoenergetic alpha-particles (energy of 3.2 MeV, LET of 124 keV microns-1) from 238Pu. The alpha-particle irradiator allows convenient irradiation of multiple dishes of cells at selectable high or low dose rates and temperatures. The survival curves of irradiated cells showed that the mean lethal dose of alpha-particles was 0.6 Gy and corresponded to an RBE, at high dose rates, of 7.9 at 80 per cent survival and 4.6 at 5 per cent survival, relative to 60Co gamma-rays. The mean areas of the 10T1/2 nuclei, perpendicular to the incident alpha-particles, was measured as 201 microns2, from which it follows that, on average, only one in six of the alpha-particle traversals through a cell nucleus is lethal. Under the well-characterized conditions of these experiments the event frequency of alpha-particle traversals through cell nuclei is 9.8 Gy-1.     

The survival of monolayers of cells growing in clusters irradiated by 211At appended to the cell surfaces.

A model of cell survival is described for the case of closely packed clusters of cells growing in monolayers. For alpha-particle decays on the cell surfaces, it is shown that cross-firing between cells produces nonuniform dose distributions within the cluster and that cells in larger clusters are exposed on average to greater doses. The model is used to simulate the survival of SK-MEL-28 human melanoma cells labeled with different radiolabeled monoclonal antibodies. The survival data suggest that this cell line is more sensitive to high-LET radiation than previously thought.     

Radiotoxicity of bismuth-213 bound to membranes of monolayer and spheroid cultures of tumor cells

Monoclonal antibody 13A to murine CD44 was used to bind the alpha-particle emitter 213Bi to cell surfaces of cultured EMT-6 or Line 1 tumor cells. Data on kinetics and saturation of binding, cell shape and nuclear size were used to calculate the absorbed dose to the nuclei. Treatment of monolayer cells with [213Bi]MAb 13A produced a classical exponential survival curve with no apparent shoulder. Microdosimetry analyses indicated that 1.4-1.7 Gy produced a 37% surviving fraction (D0). Multicellular spheroids were shown to bind [213Bi]MAb 13A mainly on the outer cell layer. Relatively small amounts of activity added to the spheroids resulted in relatively large absorbed doses. The result was that 3-6-fold less added radioisotope was necessary to kill similar fractions of cells in spheroids than in monolayer cells. These data are consistent with the interpretation that the alpha particles from a single 213Bi atom bound to one cell can penetrate and kill adjacent cells. Flow cytometry was used to sort cells originating from the periphery or from the interior of spheroids. Cells from the outside of the [213Bi]MAb 13A exposed spheroids had a lower surviving fraction per administered activity than cells from the interior. Cells were killed efficiently in spheroids up to 20-30 cells in diameter. The data support the hypothesis that alpha-particle emitters should be very efficient at killing cells in micrometastases of solid tumors.     

Comparison of the radiotoxicity of two alpha-particle-emitting immunoconjugates,terbium-149 and bismuth-213, directed against a tumor-specific,exon 9 deleted (d9) E-cadherin adhesion protein

We investigated the effects of the alpha-particle emitters (149)Tb and (213)Bi coupled to a tumor-specific antibody targeting the mutated delta 9 E-cadherin (d9 E-Cad) on single cells and cell pellets. The d9 mutation of the adhesion molecule E-cadherin is found in 10% of diffuse-type gastric cancers and is not expressed in normal tissue. Human breast cancer cells (MDA-MB-435S) transfected with d9 E-Cad or the wild-type E-cadherin gene were used to study the effects of anti-d9 E-Cad MAb coupled to (149)Tb and (213)Bi ((149)Tb-d9 MAb and (213)Bi-d9 MAb). The density of binding sites determined on transfected MDA tumor cells by Scatchard analysis and flow cytometry varied from 4 x 10(4) to 6 x 10(4) antigens per cell. Internalization of radioimmunoconjugates by cells expressing d9 E-Cad was less than 10% of bound antibody within 240 min. The effect of the radioimmunoconjugates on cell suspensions and cell pellets was quantified by [(3)H]thymidine incorporation, and the dose to the cell nuclei was determined using microdosimetric calculations. (149)Tb and (213)Bi immunoconjugates affected cells in suspension similarly. Significant differences in the proliferation capacity of d9 E-cadherin- and wild-type E-cadherin-expressing cells were observed at activity concentrations around 185 kBq/ml, corresponding to antibody concentrations between 200 ng/ml and 1000 ng/ml. Proliferation after incubation with (213)Bi-d9 MAb was 50% greater in pelleted wild-type E-Cad-expressing cells compared to wild-type E-Cad cells in suspension. In contrast, the proliferation of pelleted d9 E-Cad cells was similar to that of d9 E-Cad cells in suspension. For (149)Tb-d9 MAb, no significant difference was found between pelleted cells and cells in suspension for low activity concentrations. However, at high activity concentrations, (149)Tb-d9 MAb had only a small effect on pelleted cells. These in vitro studies demonstrate different effects of (149)Tb and (213)Bi conjugated to a tumor-specific antibody toward single cells and tumor cell pellets. Microdosimetric simulation of single cell survival after alpha-particle irradiation modeled the experimental results with reasonable accuracy.     

Simulation of binary methods for the microdosimetric analysis of cell survival after alpha-particle irradiation: ability to distinguish between different models

Analysis of cell survival after alpha-particle irradiation must account for the distribution in the amounts of energy deposited in each cell nucleus. Microdosimetric computations are usually used to determine these distributions. Irradiation with microbeams and other modern techniques has made these computations unnecessary for certain cell geometries. These techniques allow the survival of individual cells to be correlated with the amount of radiation delivered to individual cell nuclei. However, to maintain the individuality of data generated for each cell, new methods of analysis are required. In this study, we propose the use of binary methods. Each cell is regarded as a Bernoulli trial with a different probability for success (colony formation). Parameter values of the survival model are chosen to maximize the likelihood of the observed outcome. To evaluate this method, simulated data for 500, 5000 and 50,000 cells irradiated by alpha particles are analyzed along with the associated outcome for four different cell survival models. Each survival model has a different dependence on the radius of the cell nucleus. These results indicate that the model that was simulated has the highest likelihood value in all cases. However, the ability to distinguish between competing models is present only for a larger numbers of cells.     

Radioimmunotherapy with alpha-particle emitters: microdosimetry of cells with a heterogeneous antigen expression and with various diameters of cells and nuclei

Intercellular variations in the level of antigen expression and in cellular and nuclear radii were taken into account in a model used to estimate cell survival for an in vitro experiment with antibodies containing alpha-particle emitters that target the cell surface. Using measured variations in these characteristics for cells of two human cancer cell lines, the model gave results for cell survival and the fundamental parameter of radiation sensitivity, z(0), that differ substantially from those obtained using only mean values. The cell survival may be underestimated by a factor of 100 if only mean values of these cellular parameters are used, and calculated values of z(0) may be overestimated by a factor of 2. Most of this effect stems from the variation in antigen expression. The magnitudes of the differences were found to be a function of the fractions of mean specific energy delivered by surrounding activity and by activity bound to the cells.     

Relative sensitivities of repair-deficient mammalian cells for clonogenic survival after alpha-particle irradiation

The clonogenic survival of cells of the radiation-sensitive hamster cell lines irs1, irs2, irs3 and xrs5, representing different DNA repair pathways, was compared to that of their parent lines after alpha-particle irradiation. Measurements of nuclear area were made to calculate the probability of surviving a single alpha-particle traversal, the average number of lethal lesions per track and per unit dose, along with the "intrinsic radiosensitivity" of these cells, allowing for the potential of multiple lethal lesions per traversal. For all cell lines studied, alpha particles were found to be more biologically effective per unit absorbed dose than X rays at inducing cell inactivation. The repair-deficient cells showed an enhanced sensitivity to alpha particles compared to their parent line, but the degree of enhancement was less than for X rays. The reduction in additional sensitivity for alpha-particle irradiation was shown not to be due predominantly to differences in cell geometry limiting the probability of a cell nucleus being traversed. The results suggest that both the nonhomologous end-joining pathway and to a lesser extent the homologous recombination repair pathway play a role in successful repair of alpha-particle-induced damage, although a large proportion of damage is not repaired by either pathway.     

Biological response to nonuniform distributions of (210)Po in multicellular clusters

Radionuclides are distributed nonuniformly in tissue. The present work examined the impact of nonuniformities at the multicellular level on the lethal effects of (210)Po. A three-dimensional (3D) tissue culture model was used wherein V79 cells were labeled with (210)Po-citrate and mixed with unlabeled cells, and multicellular clusters were formed by centrifugation. The labeled cells were located randomly in the cluster to achieve a uniform distribution of radioactivity at the macroscopic level that was nonuniform at the multicellular level. The clusters were maintained at 10.5 degrees C for 72 h to allow alpha-particle decays to accumulate and then dismantled, and the cells were seeded for colony formation. Unlike typical survival curves for alpha particles, two-component exponential dose-response curves were observed for all three labeling conditions. Furthermore, the slopes of the survival curves for 100, 10 and 1% labeling were different. Neither the mean cluster absorbed dose nor a semi-empirical multicellular dosimetry approach could accurately predict the lethal effects of (210)Po-citrate.     

Monte Carlo modeling of the effects of injected short-, medium- and longer-range alpha-particle emitters on human marrow at various ages

The effects of injected short-, medium- and longer-range alpha-particle emitters ((149)Tb, (211)At/(211)Po and (213)Bi/(213)Po, respectively) on the total hemopoietic stem cell population of active normal bone marrow in humans of various ages has been estimated using Monte Carlo modeling. The fraction of the normal hemopoietic stem cells that are hit and survive has been calculated as a first step toward estimating the risk of development of therapy-induced leukemia. The fraction was lowest for the shorter-range alpha-particle emitter ((149)Tb) and highest for the longer-range alpha-particle emitter ((213)Bi/(213)Po), with the value for the medium-range alpha-particle emitter (211)At/(211)Po being intermediate between these. There was little variation in the data with the age of the subject within each alpha-particle emitter. This lack of age dependence provides reassurance that the fraction of cells hit in any subject of any age with normal marrow can be estimated by modeling newborn marrow (which requires little computing time) despite age-related differences in microarchitecture.     

Vascular-targeted radioimmunotherapy with the alpha-particle emitter 211At

Astatine-211, an alpha-particle emitter, was employed in a model system for vascular-targeted radioimmunotherapy of small tumors in mouse lung to compare its performance relative to other radioisotopes in the same system. Astatine-211 was coupled to the lung blood vessel-targeting monoclonal antibody 201B with N-succinimidyl N-(4-[211At]astatophenethyl) succinamate linker. Biodistribution data showed that the conjugate delivered 211At to the lung (260-418% ID/g), where it remained with a biological half-time of about 30 h. BALB/c mice bearing about 100 lung tumor colonies of EMT-6 cells, each about 2000 cells in size, were treated with 211At-labeled monoclonal antibody 201B. The administered activity of 185 kBq per animal extended the life span of treated mice over untreated controls. Injections of 370 kBq, corresponding to an absorbed dose of 25-40 Gy, were necessary to eradicate all of the lung tumors. Mice receiving 740 kBq of 211At-labeled monoclonal antibody 201B developed pulmonary fibrosis 3-4 months after treatment, as did mice treated with 3700 kBq of the alpha-particle emitter 213Bi-labeled monoclonal antibody 201B in previous work. Animals that were injected with 211At bound to untargeted IgG or to glycine, as control agents, also demonstrated therapeutic effects relative to untreated controls. Control groups that received untargeted 211At required about twice as much administered activity for effective therapy as did groups with lung-targeted radioisotope. These results were not consistent with radioisotope biodistribution and dosimetry calculations that indicated that lung-targeted 211At should be at least 10-fold more efficient for lung colony therapy than 211At bound to nontargeting controls. The data showed that 211At is useful for vascular-targeted radioimmunotherapy because lung tumor colonies were eradicated in the mice. Work in this model system demonstrates that vascular targeting of alpha-particle emitters is an efficient therapy for small perivascular tumors and may be applicable to human disease when specific targeting agents are identified.     

Hit rates and radiation doses to nuclei of bone lining cells from alpha-particle-emitting radionuclides.

Factors relating the local concentration of a bone-seeking alpha-particle emitter to the mean hit rate have been determined for nuclei of bone lining cells using a Monte Carlo procedure. Cell nuclei were approximated by oblate spheroids with dimensions and location taken from a previous histomorphometric study. The Monte Carlo simulation is applicable for planar and diffuse labels at plane or cylindrical bone surfaces. Additionally, the mean nuclear dose per hit, the dose mean per hit, the mean track segment length and its second moment, the percentage of stoppers, and the frequency distribution of the dose have been determined. Some basic features of the hit statistics for bone lining cells have been outlined, and the consequences of existing standards of radiation protection with regard to the hit frequency to cell nuclei are discussed.     

Response of X-ray-sensitive CHO mutant cells (xrs-6c) to radiation. II. Relationship between cell survival and the induction of chromosomal damage with low doses of alpha particles

The induction of cytotoxicity, chromosomal aberrations, and sister chromatid exchanges (SCEs) was measured in CHO K-1c cells and in isogenic X-ray-sensitive mutant xrs-6c cells that had been irradiated with X rays and alpha particles in isoleucine-deficient alpha-minimal essential medium in G1 phase of the cell cycle. There was a noticeable shoulder region on the survival curve for CHO K-1c cells irradiated with very low doses of alpha particles, whereas this feature was absent for xrs-6c cells with alpha-particle doses as low as 0.5 cGy. Higher frequencies of chromatid-type aberrations were induced in G1-phase xrs-6c cells than in G1-phase CHO K-1c cells by both gamma- and alpha-particle irradiation. Induction of nonlethal chromosomal aberrations was observed following exposure to 2-6 cGy of alpha particles, doses yielding 97-100% cell survival. Irradiation with 0.5 cGy of alpha particles induced SCE; nearly 60% of irradiated cells contained significantly increased levels of SCE. However, only 3% of the nuclei of cells exposed to 0.5 cGy of alpha-particle radiation were actually traversed by an alpha particle. The observation that a large fraction of cells apparently survive exposure to very low doses of alpha-particle radiation with persistent genetic damage manifested by both chromosomal aberrations and SCEs may have important implications for the carcinogenic hazards of high-LET radiation.     

The in vitro radiobiology of astatine-211 decay

Chinese hamster V79 cells in culture were exposed to astatine-211, an alpha-particle-emitting radiohalogen. The dose-log survival response was linear with no detectable shoulder. Cells in monolayers had a D0 of 1.0 microCi/ml. Suspended cells had a D0 of 0.60 microCi/ml with a cellular uptake of 2.5 fCi/cell; this is equal to approximately 1.5 alpha-particle traversals per cell nucleus. The frequencies of chromosome and chromatid breaks were linear with dose, but the number declined rapidly with time. These data are discussed in relation to published alpha-particle beam studies and the potential use of 211At in radionuclide therapy.     

Bound PCNA in nuclei of primary rat tracheal epithelial cells after exposure to very low doses of plutonium-238 alpha particles

Bystander effects from ionizing radiation have been detailed for a number of cell systems and a number of end points. We wished to use a cell culture/ex vivo rat model of respiratory tissue to determine whether a bystander effect detected in culture could also be shown in a tissue. Examination by immunofluorescence techniques of tracheal cell cultures after exposure to very low doses of alpha particles revealed a large proportion of cells with proliferating cell nuclear antigen (PCNA) bound in their nuclei. PCNA was selected as an end point because it is involved in both DNA repair and the changes in cell cycle that are typical of many reported bystander effects. Maximum response can be detected in up to 28% of the cells in sub-confluent cultures with a dose of only 2 mGy. At this dose less than 2% of the cell nuclei have experienced a particle traversal and less than 6% of the cells have experienced an alpha-particle traversal through either their nucleus or some part of their cytoplasm. The hypothesis that this bystander response in nontargeted cells is mediated through secreted factor(s) is presented, and supporting evidence was found using partial irradiation and co-culture experiments. Examination of the effect with excised pieces of trachea demonstrated a response similar to that seen in culture.     

Induction of sperm head abnormalities by incorporated radionuclides: dependence on subcellular distribution, type of radiation, dose rate, and presence of radioprotectors

In contrast to the biological effects caused by exposure to external beams of radiation, the effects of tissue-incorporated radionuclides are highly dependent on the type of radiation emitted and on their distribution at the macroscopic, microscopic, and subcellular levels, which are in turn determined by the chemical nature of the radionuclides administered. Induction of abnormalities of sperm heads in mice is investigated in this work after the injection of a variety of radiochemicals including alpha emitters. When the initial slopes of the dose-response curves are used to compare the relative biological effectiveness (RBE) of different radiocompounds, the alpha particles emitted in the decay of 210Po are more effective than Auger electrons emitted by 125I incorporated in the DNA of the spermatogonial cells, and both emissions are more effective than X rays. It is also shown that the Auger emitters (125I, 111In) distributed in the cell nucleus are more efficient in producing abnormalities than the same radionuclides localized in the cytoplasm. These findings are consistent with our earlier observations, where spermatogonial cell survival is assayed as a function of the testicular absorbed dose. Further, chronic irradiation of testis with gamma rays from intratesticularly administered 7Be is about three times more effective in causing abnormalities than a single acute exposure to 120-kVp X rays. The resulting RBE values correlate well with our data on sperm head survival with the same radiocompounds. Finally, the radioprotector cysteamine, when administered in small, nontoxic amounts, significantly reduces the incidence of sperm abnormalities from alpha-particle radiation as well as emissions from 125I incorporated into DNA, the dose reduction factors being 10 and 14, respectively.     

Distributions of cell populations within alpha-particle range of plutonium deposits in the rat and beagle testis

Plutonium is not uniformly distributed in testicular tissues; thus some cell populations may receive larger or smaller radiation exposures than would be expected if the nuclide were uniformly distributed. The distributions of cell populations within alpha-particle range of Pu deposits in rat and beagle testes were determined. The data were collected from autoradiographs of testicular tissues containing 241Pu. A cell distribution factor (CDF) was determined for each cell population and is defined as the average number of each cell type within alpha-particle range of each observed Pu deposit relative to the number of each cell type that would be expected within alpha-particle range of each Pu deposit, if the deposits were distributed uniformly. In addition, the percentage of the spermatogonial stem cell population within alpha-particle range of Pu deposits was determined. In rats, the CDF for the spermatogonial stem cells is about 2.2. This value is similar to other enhancement and inhomogeneity factors reported for rodents in the literature. In beagles the CDFs to all cells in the seminiferous epithelium were less than the rats. In addition, the percentage of spermatogonial cells within alpha-particle range of Pu concentrations in the interstitial tissues was a factor of about 3 less in the dog than in the rat. The largest CDFs seen in both species were in the interstitial tissues, particularly for Leydig cells. Because the organization of testicular tissues in the beagle is quite different from rodents but more similar to human, the results from this study suggest that extrapolations from rodents to humans may tend to overestimate the potential for radiation exposure to spermatogonial stem cells as well as the fraction of the spermatogonial stem cell population at risk to exposure from internally deposited 239Pu.     

The Adhesion GPCR CD97/ADGRE5 inhibits apoptosis

The Adhesion G protein-coupled receptor (GPCR) CD97/ADGRE5 is induced, upregulated, and/or biochemically modified in various malignancies, compared to the corresponding normal tissues. As tumor cells are generally more resistant to apoptosis, we here studied the ability of CD97 to regulate tumor cell survival under apoptotic conditions. Stable overexpression of wild-type CD97 reduced serum starvation- and staurosporine-induced intrinsic and tumor necrosis factor (TNF)/cycloheximide-induced extrinsic apoptosis, indicated by an increase in cell viability, a lower percentage of cells within the subG0/G1 phase, expressing annexin V, or having condensed nuclei, and a reduction of DNA laddering. Protection from cell death by CD97 was accompanied by an inhibition of caspase activation and modulation of anti- and pro-apoptotic members of the BCL-2 superfamily. shRNA-mediated knockdown of CD97 and, in part, truncation of the seven-span transmembrane (TM7) region of CD97 increased caspase-mediated apoptosis. Protection from apoptosis required not only the TM7 region but also cleavage of the receptor at its GPCR proteolysis site (GPS), whereas alternative splicing of its extracellular domain had no effect. Together, our data indicate a role of CD97 in tumor cell survival.     

Analysis of soft tissue tumors by an attributed minimum spanning tree.

Histologic slides of 22 soft tissue tumors (9 malignant fibrous histiocytoma, 8 fibrosarcoma, 2 rhabdomyosarcoma, 2 osteosarcoma, 1 Askin tumor) were Feulgen stained. Using an automated image analyzing system (Cambridge 570) at low magnification (25x), the tumor cell nuclei were segmented. The geometrical center of the nuclei was considered the vertex. A basic graph was constructed according to the neighborhood condition of O'Callaghan. Neighboring tumor cell nuclei were visualized by connecting edges. Several features of tumor cell nuclei were measured, including area, surface, major and minor axis of best fitting ellipsis and extinction (DNA content). Nuclear features are attributed to the vertices. The differences, or "distances," between features of connected vertices are attributed to the corresponding edges, which are dependent on the attributes. Thus, different minimum spanning trees (MST) result. Each MST can be decomposed into clusters using a suitable decomposition function on the edges, which rejects an edge if its attributes differ from the mean of the attributed values of surrounding edges more than a neighbor dependent bound (lower limit). Taking into account the length and other attributes of edges (e.g., differences in orientation of the major axis), clusters of different nuclear orientation can be detected. A cluster tree can be constructed by defining the geometric center of a cluster as a new vertex, and by computing the neighborhood of the cluster vertices. The result is an attributed MST containing characteristic structural properties of the image (in cases of sarcomatous tumors, local orientation of tumor cell nuclei and local DNA abnormalities).     

Water transport in a cluster of closely packed erythrocytes at subzero temperatures

A one-dimensional model has been developed to describe the kinetics of water transport in a cluster of closely packed cells. For the case of human red blood cells, the intracellular medium has been treated as an ideal, hydrated, nondilute multicomponent electrolyte solution. Results show that the volume flux of water out of the interior cells of the cluster lags behind that of the exterior cells. At any given temperature (or time), the amount of water retained within a cluster of closely packed cells of a given type exceeds (on an overall percentage basis) the amount of water retained within a single isolated cell of the same type. For a given cooling rate the probability of intracellular ice nucleation at any given temperature will therefore be greater for cells in the interior of a cluster, and the survival signature for a cell cluster should peak at a cooling rate which is less than the corresponding optimal value for a single, isolated cell. These results are consistent with experimental observations.     

Enhanced loading efficiency and retention of 225Ac in rigid liposomes for potential targeted therapy of micrometastases

Targeted alpha-particle emitters are promising therapeutics for micrometastatic disease. Actinium-225 has a 10-day half-life and generates a total of four alpha-particles per parent decay rendering (225)Ac an attractive candidate for alpha-therapy. For cancer cells with low surface expression levels of molecular targets, targeting strategies of (225)Ac using radiolabeled carriers of low specific radioactivities (such as antibodies) may not deliver enough alpha-particle emitters at the targeted cancer cells to result in killing. We previously proposed and showed using passive (225)Ac entrapment that liposomes can stably retain encapsulated (225)Ac for long time periods, and that antibody-conjugated liposomes (immunoliposomes) with encapsulated (225)Ac can specifically target and become internalized by cancer cells. However, to enable therapeutic use of (225)Ac-containing liposomes, high activities of (225)Ac need to be stably encapsulated into liposomes. In this study, various conditions for active loading of (225)Ac in preformed liposomes (ionophore-type, encapsulated buffer solution, and loading time) were evaluated, and liposomes with up to 73 +/- 9% of the initial activity of (225)Ac (0.2-200 microCi) were developed. Retention of radioactive contents by liposomes was evaluated at 37 degrees C in phosphate buffer and in serum-supplemented media. The main fraction of released (225)Ac from liposomes occurs within the first two hours of incubation. Beyond this two hour point, the encapsulated radioactivity is released from liposomes slowly with an approximate half-life of the order of several days. In some cases, after 30 days, (225)Ac retention as high as 81 +/- 7% of the initially encapsulated radioactivity was achieved. The (225)Ac loading protocol was also applied to immunoliposome loading without significant loss of targeting efficacy. Liposomes with surface-conjugated antibodies that are loaded with (225)Ac overcome the limitations of low specific activity for molecular carriers and are expected to be therapeutically useful against tumor cells having a low antigen density.