131I-anticarcinoembryonic antigen therapy of LS174T human colon adenocarcinoma spheroids.

LS174T human colon adenocarcinoma multicell spheroids were used to study the radiobiological aspects of radioimmunotherapy. The spheroids were incubated in 131I-anticarcinoembryonic antigen (B7) at an antibody concentration of 0.5 microgram/ml and at 131I concentrations of 2.5 and 7.5 microCi/ml. After incubation times of 90 h, clonogenic cells per spheroid were reduced by 1400-fold and 23-fold at the high and low 131I concentrations, respectively. 131I Nonspecific antibody (PX63) resulted in 2- and 1.2-fold reductions. Spheroid diameter was not significantly affected by therapy but histological examination revealed that there had been a significant reduction in the cell density, particularly near the spheroid surface. Using a theoretical model to estimate radiation dose, a radiation survival curve was constructed. The resulting curve was somewhat concave suggesting the presence of a resistant population of cells. It is likely that this observation is primarily due to the fact that the inner cells received a lower dose than the outer cells. A population of radiobiologically hypoxic cells in the inner portion of the spheroids may also have contributed to the decreasing slope of the curve as well as ongoing cell division leading to new cells which receive a lower radiation dose per cell cycle. Because of the ability to estimate radiation dose for a given biological effect, these types of experiments may allow predictions of the efficacy of radiolabeled antibody therapy for micrometastatic disease.

Erythropoietin-receptor expression and function during the initiation of murine yolk sac erythropoiesis.

Although erythropoietin is necessary for definitive (fetal liver and bone marrow) erythropoiesis, the role of erythropoietin signaling in primitive (yolk sac) hematopoiesis has not been well defined. In situ hybridization studies have revealed that erythropoietin-receptor (EPOR) mRNA accumulation begins in mesoderm cell masses of the developing yolk sac of the neural plate stage embryo (E7.5) before the development of morphologically recognizable erythroblasts. EPOR mRNA is also present in yolk sac blood islands at early somite stages (E8.5). These findings suggest that EPOR functions during early stages of yolk sac erythropoiesis. We have used a serum-free murine yolk sac explant system (Palis et al., Blood 86:156, 1995) to investigate the initial differentiation of primitive erythroblasts from extraembryonic mesoderm cells. Exogenous erythropoietin increased both erythroblast numbers and betaH1-globin accumulation in yolk sac explants, suggesting that primary yolk sac erythroblasts are directly responsive to erythropoietin. An antisense oligodeoxynucleotide (ODN) experimental approach was used to examine the functional role of erythropoietin signaling during the initiation of yolk sac hematopoiesis in yolk sac explants. Antisense EPOR ODN produced a >50% reduction (p < 0.005) in the number of differentiating primitive erythroblasts, >95% reduction in betaH1-globin accumulation (p < 0.001), and a >50% reduction (p < 0.01) in the number of CFU-E and BFU-E compared with missense EPOR ODN-treated and untreated control explants. Antisense EPOR ODN also blocked the increase in primitive erythroblast number induced by exogenous erythropoietin. We conclude that erythropoietin/EPOR signaling is functionally active during the initial proliferation and differentiation of primary yolk sac erythroblasts. These results also suggest that other growth factor signaling cascades are active during the onset of mammalian erythropoiesis.

The hypoxic cytotoxin SR 4233 increases the effectiveness of radioimmunotherapy in mice with human non-Hodgkin's lymphoma xenografts.

PURPOSE: To determine if either the hypoxic cell radiosensitizer etanidazole (SR 2508) or the hypoxic cytotoxin SR 4233 could improve the effectiveness of radioimmunotherapy. METHODS AND MATERIALS: LC4 (an IgG1 monoclonal antibody directed toward malignant T cells) and MB-1 (an irrelevant isotype-matched control antibody) were injected intraperitoneally into severe combined immunodeficient phenotype mice with human cutaneous T cell lymphoma xenografts in order to determine the distribution of the antibodies in the tumors and normal tissues as a function of time. Computerized-pO2-histography was used to measure the median oxygen tension in the tumors. Tumor-bearing mice were treated with: (a) LC4; (b) 90Y-LC4; (c) 90Y-MB-1; (d) whole body irradiation delivered via an external 137Cs source; (e) etanidazole and 90Y-LC4; (f) SR 4233 and 90Y-LC4; (g) etanidazole; and (h) SR 4233. An additional group of mice received no treatment and served as controls. A tumor growth delay assay was used to assess the effectiveness of the different treatment regimens. RESULTS: LC4 accumulated in the tumors to a significantly greater extent than MB-1 (p < 0.001) and reached a peak concentration in the tumors 5 days post-injection. The human cutaneous T cell lymphoma xenografts had a relatively low median oxygen tension. LC4 by itself was able to produce a minor decrease in tumor size (control vs. LC4; p = 0.001). 90Y-LC4 produced greater tumor growth delay than LC4 alone (LC4 vs. 90Y-LC4; p = 0.01); however, the Yttrium-90 caused neutropenia and weight loss. The 90Y-labeled tumor-specific and non-specific antibodies both exerted greater tumor growth delay than externally delivered whole body irradiation (p < or = 0.03) due to preferential uptake of the antibodies in the tumors. Etanidazole and SR 4233 by themselves did not significantly inhibit the growth of the tumors. Etanidazole did not significantly enhance the tumor growth delay produced by 90Y-LC4 (90Y-LC4 vs etanidazole and 90Y-LC4, p = 0.13). SR 4233, on the other hand, did enhance the tumor growth delay produced by 90Y-LC4 (90Y-LC4 vs. SR 4233 and 90Y-LC4, p = 0.046). The neutropenia and weight loss caused by 90Y-LC4 were exacerbated slightly (< 10%) by the administration of SR 4233. CONCLUSIONS: A first generation hypoxic cytotoxin, SR 4233, was able to enhance the tumor growth delay produced by radioimmunotherapy in severe combined immunodeficient phenotype mice with human cutaneous T cell lymphoma xenografts.

The effect of antigen concentration, antibody valency and size, and tumor architecture on antibody binding in multicell spheroids.

Intact IgG1 and F(ab')2 anti-carcinoembryonic antigen antibodies penetrate human colon adenocarcinoma multicell spheroids much more slowly than Fab fragments and the molecular weight and the binding site valency appear to be the most important factor in determining the rate of penetration. The rate is also influenced considerably by the number of antigen binding sites per cell, with a high antigen concentration slowing penetration appreciably. The tumor cell architecture appears to have a minor effect on antibody penetration when compared to antibody size or antigen concentration.

Embryonic expression and function of the chemokine SDF-1 and its receptor, CXCR4.

Directed cell movement is integral to both embryogenesis and hematopoiesis. In the adult, the chemokine family of secreted proteins signals migration of hematopoietic cells through G-coupled chemokine receptors. We detected embryonic expression of chemokine receptor messages by RT-PCR with degenerate primers at embryonic day 7.5 (E7.5) or by RNase protection analyses of E8.5 and E12.5 tissues. In all samples, the message encoding CXCR4 was the predominate chemokine receptor detected, particularly at earlier times (E7.5 and E8.5). Other chemokine receptor messages (CCR1, CCR4, CCR5, CCR2, and CXCR2) were found in E12.5 tissues concordant temporally and spatially with definitive (adult-like) hematopoiesis. Expression of CXCR4 was compared with that of its only known ligand, stromal cell-derived factor-1 (SDF-1), by in situ hybridization. During organogenesis, these genes have dynamic and complementary expression patterns particularly in the developing neuronal, cardiac, vascular, hematopoietic, and craniofacial systems. Defects in the first four of these systems have been reported in CXCR4- and SDF-1-deficient mice. Our studies suggest new potential mechanisms for some of these defects as well as additional roles beyond the scope of the reported abnormalities. Earlier in development, expression of these genes correlates with migration during gastrulation. Migrating cells (mesoderm and definitive endoderm) contain CXCR4 message while embryonic ectoderm cells express SDF-1. Functional SDF-1 signaling in midgastrula cells as well as E12.5 hematopoietic progenitors was demonstrated by migration assays. Migration occurred with an optimum dose similar to that found for adult hematopoietic cells and was dependent on the presence of SDF-1 in a gradient. This work suggests roles for chemokine signaling in multiple embryogenic events.