Comparison of sensitivities of cells to X-ray therapy, chemotherapy, and isotope therapy using a tumor spheroid model.

Numerous treatment modalities have been used in treating microscopic carcinoma confined to the peritoneal cavity. However, there is little information at the cellular level regarding which modality is most effective in eradicating small nests of tumor cells. In order to determine whether chemotherapy, X rays, or isotope therapy is most effective, we have compared in a three-dimensional spheroid model the survival of cells after treatment with cis-platinum, X ray, or the investigational high-energy isotope 212-bismuth (Bi-212). In this study, V-79 cells were grown to 100-microns diameter spheroids. In killing spheroids, high-energy isotopes were at least six times more effective than X rays at fractionated doses of up to 1300 cGy. High-energy isotopes appear also to be as efficient as cis-platinum. Regardless of exposure time to treatment, Bi-212 was more toxic to spheroids than the other treatment modalities. From this study we conclude that high-energy isotope therapy biologically is very effective in eradicating microscopic nests of cells.

Morphological, biochemical, and molecular changes in endothelial cells after alpha-particle irradiation.

The response of cultured bovine aortic endothelial (BAE) cells after exposure to alpha-particle radiation from chelated 212Bi has been evaluated. The results suggest that even relatively high doses of alpha-particle radiation from 212Bi (20-72 Gy) cause only minor acute changes in the morphology of BAE cells (light and electron microscopy) under conditions of confluent monolayer growth. Significant morphological changes can be detected in cells that detach from the monolayer, though it is unclear whether these changes represent a genuine response to irradiation or reflect the causes or effects of monolayer detachment with the consequent loss of intercellular biochemical communication. After alpha-particle irradiation (20-40 Gy) angiotensin-converting-enzyme activity was not detectable in the monolayer culture medium but was significantly decreased within the cell monolayer. Neutral-elution-assay data demonstrated that DNA double-strand-break (DSB) damage occurred in these cells and that about 35% of the DSBs were repairable.

Preparation and evaluation of para-[211At]astatobenzoyl labeled anti-renal cell carcinoma antibody A6H F(ab')2. In vivo distribution comparison with para-[125I]iodobenzoyl labeled A6H F(ab')2.

A preliminary investigation of an 211At labeled anti-renal cell carcinoma antibody fragment, A6H F(ab')2, was conducted. In the investigation, A6H F(ab')2 was labeled by conjugation with N-succinimidyl p-[211At]astatobenzoate, and the in vivo biodistribution was evaluated in athymic mice bearing TK-82 renal cell carcinoma xenografts. As a control, p-[125I]iodobenzoyl labeled A6H F(ab')2 was coinjected with the astatinated F(ab')2. The data obtained demonstrated that the two radiolabels (211At and 125I) had quite similar distributions, providing evidence that the 211At remained attached to the A6H F(ab')2 in vivo. Further, the astatinated antibody attained a 2:1 tumor-to-blood ratio, and greater than 35:1 tumor-to-muscle ratio, at 4h post-injection, suggesting that this antibody conjugate could be used to evaluate treatment of metastatic renal cell carcinoma in a mouse model.

G2M arrest and apoptosis in murine T lymphoma cells following exposure to 212Bi alpha particle irradiation.

Asynchronous exponentially growing EL4 murine T lymphoma cells were exposed either to high LET alpha-radiation from 212Bi-DTPA or to gamma-radiation from a 137Cs source. Radiation-induced cell cycle perturbation was studied by flow cytometry. Alpha irradiation, like gamma, transiently arrested cells in the G2M phase in a dose-dependent manner. The maximum percentages of cells accumulated in G2M 18 h after alpha- and gamma-irradiation were comparable, though the dose-response relationships differed. The "RBE" value for G2M block for alpha- versus gamma-radiation was approx. 4. Electron microscopic studies of the cell samples where a large proportion of cells were arrested in G2M showed subcellular changes in nuclear membrane and the presence of morphologically apoptotic cells. Biochemical analysis of DNA from irradiated cells by agarose gel electrophoresis revealed more extensive DNA fragmentation for alpha- vs gamma-irradiation, even at relatively low total doses. We conclude that the high LET radiation is more efficient in inducing G2M block and apoptosis in EL4 lymphoma cells. The overall radiosensitivity of some high and low grade malignant lymphoma cells to radiation may correlate with these processes. The clinical implications of 212Bi-induced G2M delay may be particularly important for biologically targeted high LET radiopharmaceutical therapy.

Localization of sarcoma xenografts in nude mice with indium-111-labeled monoclonal antibodies.

A labeling method utilizing modified carbohydrate moieties in antibody heavy chains as radionucleotide binding sites was evaluated. Murine anti-sarcoma monoclonal antibody (MAb 19-24) was labeled with Indium-111 (111In) using this technique and subcutaneous human sarcoma xenografts were successfully localized in nude mice. A nonspecific monoclonal antibody BL-3 was used as a negative control. Tumor-to-blood ratios of radioactivity in the mice injected with 111In-labeled MAb 19-24 were significantly (P < 0.05) higher than those obtained with nonspecific MAb BL-3. Calculations of percentage injected dose of radioactivity per gram tissue showed relatively high specific uptake of MAb 19-24 in sarcoma xenografts. Radioactivity cleared from the blood rapidly and hepatic uptake of 111In-labeled antibodies was found to be relatively low. Biodistribution studies in normal mice with 111In-labeled antibodies showed only blood pool activity with no significant concentration of activity into organs. Therefore, immunoreactivity of the antibodies was retained after 111In-labeling utilizing this new technique, allowing specific binding of radiolabeled MAb to tumor xenografts with relatively low hepatic uptake.

Induction of mutations by bismuth-212 alpha particles at two genetic loci in human B-lymphoblasts.

The human lymphoblast cell line TK6 was exposed to the alpha-particle-emitting radon daughter 212Bi by adding DTPA-chelated 212Bi directly to the cell suspension. Cytotoxicity and mutagenicity at two genetic loci were measured, and the molecular nature of mutant clones was studied by Southern blot analysis. Induced mutant fractions were 2.5 x 10(-5)/Gy at the hprt locus and 3.75 x 10(-5)/Gy at the tk locus. Molecular analysis of HPRT- mutant DNAs showed a high frequency (69%) of clones with partial or full deletions of the hprt gene among radiation-induced mutants compared with spontaneous mutants (31%). Chi-squared analyses of mutational spectra show a significant difference (P < or = 0.005) between spontaneous mutants and alpha-particle-induced mutants. Comparison with published studies of accelerator-produced heavy-ion exposures of TK6 cells indicates that the induction of mutations at the hprt locus, and perhaps a subset of mutations at the tk locus, is a simple linear function of particle fluence regardless of the ion species or its LET.

Interlaboratory comparison of different alpha-particle and radon sources: cell survival and relative biological effectiveness.

Alpha radiation-induced cell killing was determined in four different laboratories in order to: 1) measure interlaboratory variability and 2) compare the effects of radon and radon daughter exposures with the effects of 238Pu (an often-used model for radon exposure). The results suggest that differences in handling from laboratory to laboratory can affect both low and high linear energy transfer responses and should be considered when comparing results from different laboratories.

Cellular kinetics, dosimetry, and radiobiology of alpha-particle radioimmunotherapy: induction of apoptosis.

Though clinical results for radioimmunoconjugate therapy of most common epithelial tumors have been disappointing, dramatic responses have been observed repeatedly in the treatment of high- and low-grade malignant lymphomas. This high clinical responsiveness after radioimmunoconjugate therapy sometimes appears to be out of proportion to the calculated radiation dose absorbed by the lymphoma tissue. Here we describe some key aspects of the kinetics, dosimetry, and cellular radiobiology of murine lymphoma cells exposed to 212Bi-radiolabeled alpha-particle-emitting immunoconjugates specific for the differentiation antigen Thy 1.2. Approximately 25 cell-bound alpha-particle-emitting immunoconjugates per target cell were required to reduce clonogenic survival by 90% (the radiobiological D10). Serial kinetic analyses of the antibody and radioisotope components of the immunoconjugates revealed significant levels of dechelation and up to 7.5% cellular internalization of the isotope. Cellular radiation dosimetry performed by Monte Carlo computer simulation of alpha-particle energy deposition patterns based on the observed radiopharmacokinetics showed that the D10 resulted from approximately four alpha-particle traversals through the nucleus, corresponding to an absorbed radiation dose of approximately 0.95 Gy to the cell nucleus. Electron micrographs and DNA gel studies of murine lymphoma cells undergoing radioimmunoconjugate therapy in vivo and in vitro demonstrated bizarre blebbing patterns, condensation of chromosomal material, and internucleosomal DNA fragmentation patterns characteristic of programmed cell death (apoptosis). We conjecture that the efficacy of radioimmunoconjugates against responsive cell types may be the result of passive DNA damage by ionizing radiation and the initiation of apoptosis in response to radioimmunotherapy.

Long-term biodistribution in tumored mice of murine and chimeric B72.3-IgG antibody radiolabeled with 114mIn via both DTPA and a macrocyclic chelator.

To assess the possibilities of using 114mIn as a therapeutic agent, the long-term biodistribution of 114mIn was studied, in tumor-bearing nude mice, after injection of labeled monoclonal antibody (MoAb) B72.3 IgG, either DTPA-coupled murine, DTPA-coupled chimeric, or macrocycle-coupled chimeric antibody. Although the biodistributions in all cases were similar, there were important differences. The use of DTPA-coupled chimeric antibody led to higher concentrations of radioactivity in tumor, and to lower concentrations in liver and bone, as compared to DTPA-coupled murine antibody. The use of macrocycle-coupled chimeric antibody led to higher concentrations of radioactivity in the liver and in bone as compared to the DTPA-coupled chimeric antibody. However, in this case there were no significant differences in tumor uptake or clearance. Radiation doses were calculated based on the organ retention and by neglecting source-to-target contributions. Radiation dose distribution was marginally favorable for therapy in the group injected with DTPA-coupled chimeric antibody.

Analysis of ascites from patients with ovarian carcinoma by cell flow cytometry.

Cell flow cytometry offers the opportunity to analyze cytopathological samples with regards to DNA content and proliferative activity. To investigate whether this modality can quantitate certain aspects of ovarian carcinoma by analyzing ascites, 43 samples from patients with advanced papillary serous adenocarcinoma of the ovary were studied. In 28 samples (65%) ploidy and the percentage of cells in S phase (%S phase) could be analyzed. Fifteen samples could not be analyzed because of overlapping cell populations distorting distinct cell cycle phases. Of the 28 samples studied, 8 (29%) were diploid and 20 (71%) were aneuploid. The DNA in aneuploid samples ranged from 1.23 to 2.65. The %S phase for aneuploid was greater than that for diploid samples. Patients with diploid samples survived longer. Cytometric analysis of cells from ascites in 4 patients in whom disease progressed after they received chemotherapy showed that the percentage of cells in S phase increased. Cells from ascites established in vitro showed that ploidy and proliferative activity changed as cells were passed in culture. In conclusion, the analysis of ascites by cell flow cytometry may be a prognosticator in patients with advanced ovarian carcinoma. In addition, conclusions extrapolated from in vitro data to the in vivo situation should be done cautiously since late-passaged cells may not always be representative of the initial tumor sample.

Accumulation of indium-111-labeled human low density lipoprotein in the rabbit aorta: Implications for nuclear imaging of vascular lesions.

Nuclear imaging of atheromata must distinguish lesions from both blood pool and normal arterial tissue. We have examined spatial and temporal variations of indium-111-labeled human low density lipoprotein (LDL) accumulation in rabbit aortas. LDL-derived In-111 activity was time-independent in lesion-resistant regions of aortas from normal and hypercholesterolemic animals (mean 2.9 × 10(-6) percent injected activity per milligram tissue [%IA/mg]) and in lesion-prone regions of normal aortas (mean 7.1 × 10(-6) %IA/mg). In contrast, activity in sudanophilic lesions of hypercholesterolemic rabbit aortas reached a peak of 31 × 10(-6) %IA/mg at 92 hours postinjection. The mean ratio between activity in lesions versus lesion-resistant regions described a broad convex curve with minima of 4:1 at 14 hours and 136 hours and a peak of 14:1 measured at 72 hours postinjection. The mean ratio between In-111 in lesions and blood followed a sigmoid curve, rising exponentially from 1:25 at 14 hours to 1:3 by 72 hours postinjection. We conclude that optimal signal-to-noise ratios for monitoring atheroma-associated LDL-derived radioactivity occur late, not before about 3 days postinjection. Therefore, LDL labeled with In-111 or even longer-lived radionuclides holds the greatest promise for effective clinical nuclear imaging of atherosclerosis.

Differential locus sensitivity to mutation induction by ionizing radiations of different LETs in Chinese hamster ovary K1 cells.

Mutation induction after exposures to 250 kVp X-rays, alpha-particles from the radon daughter 212Bi, and fission-spectrum neutrons from the JANUS reactor was studied in Chinese hamster ovary (CHO) K1 cells and in CHO-10T5, a K1 derivative containing the bacterial gene xanthine-guanine phosphoribosyl transferase (gpt). Mutation induction was analyzed at three genetic loci: the gpt locus, the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus, and the thymidine kinase (tk) locus. After X-irradiation, mutants were induced at the tk loci at approximately 8-9 times the rate of mutant induction at the hprt locus, and the rate of mutant induction at the gpt locus was 8-10 times greater than that at the hprt locus. Neutron and alpha-radiation were more effective mutagenic agents. Mutant frequencies were approximately 4- to 6-fold higher than for X-rays at the hprt and gpt loci and greater than 12-fold greater than X-rays at the tk locus. The greater sensitivity of the tk locus to mutation induction by ionizing radiation (especially neutron and alpha-particle radiation) compared to the hprt locus is likely to be due to the recovery of an additional class of mutants, possibly ones containing larger-sized mutational events. Approximately half of the X-ray-induced tk-1- mutants were small-colony mutants, and 75% of the alpha- and neutron-induced tk-1- mutants were small-colony mutants. The increase in the proportion of small-colony mutants seen with increasing radiation linear energy transfer (LET) suggests that the radiation quality influenced the type of mutation recovered at this locus. There is probably a different reason for the hypersensitivity of the gpt locus because the frequency of gpt mutants, compared to the hprt locus, was independent of radiation quality. Therefore, the LET dependence of mutant induction is gene specific and not necessarily related to the size of deletion recoverable.

In vitro exposure of mammalian cells to radon: dosimetric considerations.

We have developed a model to calculate the dose to the cell nucleus in cells exposed in suspension to radon and/or radon progeny. The model addresses the influence of (1) different radiation qualities and energies in the irradiation milieu; (2) the contribution to dose from radioactivity in the medium surrounding the cell after exposure to the radon gas as well as that from excess radon progeny associated with the cell; (3) the geometry of the cell and of the radiosensitive target, the cell nucleus; (4) the intracellular localization of the radionuclides; (5) attenuation of the alpha particles by the cytoplasm; (6) the radionuclide concentrations in the medium; and (7) the length of exposure. Investigation of the influence of these various parameters was made using an irradiation system in which cells were exposed to 212Bi, which decays to stability with the emission of an alpha particle (either 6.05 or 8.78 MeV). The information from these studies was then used to develop the system further for more complex systems in which 222Rn and its progeny are present. The model takes into account the contribution of dose from different radiation sources using scintillation counts of the medium and the cells, and it is useful for calculations of dose in situations where cells are exposed in suspension culture.

Astatine-211 labeling of an antimelanoma antibody and its Fab fragment using N-succinimidyl p-astatobenzoate: comparisons in vivo with the p-[125I]iodobenzoyl conjugate.

Astatine-211 labeling of an antimelanoma antibody, NR-ML-05, and its Fab fragment with N-succinimidyl p-[211At]astatobenzoate (2a) has been described. Preparation of the astatinated intermediate 2a was accomplished by distilling astatine-211 from an irradiated bismuth target directly into a reaction mixture containing an organometallic compound, N-succinimidyl p-(tri-n-butylstannyl)benzoate (1), and an oxidant, N-chlorosuccinimide, in 5% HOAc/MeOH. Trapping of distilled astatine as 2a was found to be efficient, resulting in 70-90% yields based on the amount of astatine-211 in the reaction mixture. The dry distillation technique employed gave recoveries of astatine-211 which ranged from 20% to 75%. Conjugation of 2a to NR-ML-05 and its Fab fragment was accomplished in 40-60% yields. The [211At]astatobenzoyl-conjugated antibodies were found to be stable in vitro when challenged by strong denaturants and nucleophilic reagents. Coinjected dual-labeled studies of the 2a astatinated antibodies and the same antibodies labeled with N-succinimidyl p-[125I]iodobenzoate (2b) in athymic mice bearing the human tumor xenograft A375 Met/Mix demonstrated that both radiolabeled antibodies had equivalent tumor localization. Data from the dual-labeled biodistribution of the intact antibody suggests that the astatine is stably attached. Data from the dual-labeled Fab fragment suggests that a portion of the astatine label is released as astatide, either from the astatinated Fab or from a catabolite.

The effects of radon daughter alpha-particle irradiation in K1 and xrs-5 CHO cell lines.

We investigated the radiobiological effects of the radon daughter bismuth-212 (212Bi) in Chinese hamster ovary (CHO) K1 cells and in xrs-5 cells, which are X-ray sensitive and deficient in the ability to rejoin DNA double-strand breaks. The cells were exposed to 250 kVp X-rays or to 212Bi chelated to diethylene triamine pentaacetic acid (DTPA); chelation of 212Bi to DTPA prevented its attachment to or entry into the cells. Cytotoxic, clastogenic, and mutagenic responses of the cells were measured and RBEs (D10, 2 chromatid aberrations/cell and 10 induced 6-thioguanine-resistant mutants) were calculated to be 3.8, 3.5, and 3.9, respectively for K1, and 1.4, 0.8, and 5.1, respectively, for xrs-5. With the exception of the RBE of less than 1 for alpha-induced aberrations in xrs-5, the results are consistent with the following conclusions: (1) alpha-particles are in general more effective cytotoxic, clastogenic and mutagenic agents than X-rays; (2) the primary lethal and clastogenic lesion induced by both X-rays and alpha-particles is probably a DNA double-strand break; (3) DNA double-strand breaks induced by alpha-radiation are less well repaired than those induced by X-rays, although a portion of alpha-induced damage is repairable; and (4) deficiencies in rejoining DNA double-strand breaks affect the clastogenic and cytotoxic effects of X-rays and alpha-radiation, not their mutagenic effects. The RBE of 0.8 for aberration induction in xrs-5 cells could reflect a deficiency in the ability of these cells to convert alpha-induced damage to chromosome aberrations. Alternatively, the RBE of less than 1 might reflect an unusual sensitivity of xrs-5 cells to alpha-induced G2 delays.

Increased nuclear damage by high linear energy transfer radioisotopes applicable for radiodirected therapy against radiologic malignancies.

High linear energy transfer radioisotopes carried by appropriate agents have been proposed for receptor-directed radiotherapy. Two such classes of isotopes are Auger electron and alpha-emitting nuclides. To determine the relative cytotoxicity and nuclear damage to cells produced by these two classes of nuclides, we compared bromine-80m (80mBr), an Auger-electron-emitting radionuclide with a 4.4-hour half-life, with bismuth-212 (212Bi), an alpha-emitter with a 1-hour half-life. Because of the short path length of the Auger electrons, 80mBr was radiotoxic only when incorporated into DNA, such as in the form of [80mBr]bromodeoxyuridine ([80mBr]BrUdR). Both agents induced linear increases in chromosome aberration frequency, however, [80mBr]BrUdR caused multiple aberrations including the shattering of parts of the chromosomes. While, in contrast, a 2-hour exposure of cells to 212Bi, chelated to DTPA, a form which does not enter the cell, induced much less extensive chromosome damage. Exposure to equivalent activities of Auger electrons or alpha-particles results in 5 times more damage in Auger-electron-exposed cells. However, estimates of dose suggest they are equally toxic. Unlike Auger electrons, alpha-particles did not need to be in as close proximity to the DNA to have clastogenic and radiotoxic effects.

Modeling of dose to tumor and normal tissue from intraperitoneal radioimmunotherapy with alpha and beta emitters.

Dose distributions for normal and tumor tissues from intraperitoneally administered radiolabeled antibodies have been calculated for 90-Yttrium (90Y), 131-Iodine (131I), and 211-Astatine (211At). The dose calculations use data on the activity of intraperitoneal fluid administered, the percent injected dose/gm uptake by tumor, biological half life, and a model for diffusion of antibody/radionuclide complex into peritoneal tissues. Calculations are performed for planar and hemispherical tumor shapes, ranging in size to establish the influence of geometry on dose distribution. Calculations for tumor geometry obtained from biopsies are also performed. When the activity is concentrated on or near the tumor surface, the maximum dose to a planar tumor for a 20 mci administration of 90Y is approximately 60 Gy, and falls rapidly to 50% of this value within 1 mm. However, for a hemispherical tumor, the dose is a maximum of 26 Gy, with an average of approximately 20 Gy. The surface dose from 131I (130 mci) is 240 Gy, and diminishes to 20 Gy in .05 cm in the planar case, whereas a hemispherical tumor receives a dose of 90 Gy over a large fraction of the volume, with the distal portions receiving 40 Gy. The surface dose for an administration of 70 mci of 211 At is 450 Gy and decreases to 50% of this value in 30 microns. Both surface geometry and tumor size are important determinants in the heterogeneity of tumor dose, as are the dose administered, antibody uptake, biodistribution, and residence time factors. These initial studies suggest that the size of disease which may be effectively treated is much less than the range of the particle emitted by radiolabeled antibodies. Furthermore, therapy is ultimately limited by the degree to which the antibody/radionuclide complex can diffuse and permeate the tumor.

Iodine-125-NRLU-10 kinetic studies and bismuth-212-NRLU-10 toxicity in LS174T multicell spheroids.

Alpha emitter-labeled antibodies (Abs) are of considerable interest in cancer therapy. Alpha particles are densely ionizing and therefore have a high radiobiologic effectiveness, and the cell killing produced is influenced very little by dose rate or hypoxic conditions. LS174T human colon adenocarcinoma spheroids were used in this study to evaluate the efficacy of alpha emitter-labeled Abs in a three-dimensional model. NRLU-10, an IgG2b Ab to a pancarcinoma antigen, and its Fab fragment were used. Initial kinetic studies using 125I-NRLU-10 revealed that a large number of binding sites/cell and high Ab affinity led to slow Ab penetration. This effect could be overcome by increasing the Ab concentration ten-fold for Fab but not for intact Ab. Bismuth-212-NRLU-10 therapy was very effective in killing single cells (over 3 log reduction in surviving fraction) but was ineffective in spheroids (less than 1 log reduction). This was likely due to inadequate penetration into the spheroids before the 212Bi decayed. The use of higher Ab concentrations, tumors with fewer antigenic sites/cell for the Ab being used, lower affinity Abs, alpha emitters with longer half-lives, and pretargeting with bifunctional Ab are all potential ways of increasing the efficacy of alpha emitter-labeled Abs for cancer therapy.