Testing prospective anticancer drugs against human tumors using simple in vivo models.

The activities of established and experimental antitumor drugs were tested against the BRO and MeWo human melanomas transplanted in the subrenal capsule of phenotypically immunocompetent (CBA x C57BL/6)F1 mice. Immunosuppression was achieved with preliminary whole-body radiation, and cytological examination showed that the tumors of untreated mice consisted largely of viable tumor cells. Several drugs tested showed moderate activity against one or both tumors. The system described provides a basis for testing anticancer agents against a panel of human melanomas implanted in immunocompetent mice.

[The selective toxicity of 5-hydroxymethyldeoxyuridine for the cells of human adenocarcinoma of the large intestine]. / Selektivnaia toksichnost' 5-gidroksimetildezoksiuridina dlia kletok adenokartsinomy tolstoi kishki cheloveka.

Comparing the toxicities of potential anticancer agents for tumorous and normal cells derived from human intestinal epithelium may be a preferred approach for in vitro testing of compounds directed against colon carcinoma. 5-Hydroxymethyldeoxyuridine, a thymidine analog, was preferentially cytotoxic for two lines of human colon adenocarcinoma cells compared to a cell line derived from normal human fetal intestine. Unlike deoxyuridine and deoxycytydine, thymidine protected HT-29 human adenocarcinoma against 5-hydroxymethyldeoxyuridine toxicity, suggesting that thymidilate synthetase is the probable target enzyme of this thymidine analog. 5-Hydroxymethyldeoxyuridine may hold promise as an agent with specific activity against human adenocarcinoma cells.

Lethality of human melanoma cells for normal mice immunosuppressed with cyclosporin A.

After intraperitoneal inoculation of BRO human melanoma cells, phenotypically immunocompetent mice given cyclosporin A (CyA) developed extensive and lethal tumors. Survivals were not significantly different for normal mice treated with this immunosuppressant compared to nude mice, which lack functional T-lymphocytes. BRO cells could be passaged in CyA-treated mice without alteration of isozymes or other properties tested. This model may have implications for growth and metastasis of human tumors under immunosuppressed conditions and may be useful for studying the mechanism of action of CyA in vivo.

A simple and sensitive method to measure radiolabeled antibody uptake by tumors in nude mice.

A method for detecting the uptake of 125I-labeled monoclonal antibody by human tumors xenografted into nude mice is described. After implantation of the tumor intramuscularly in one of the forelimbs, radioactivity was measured in the area of tumor inoculation and in the contralateral normal forelimb. For these measurements, a lead apparatus was constructed which shielded over 99% of the whole body radioactivity and permitted efficient counting of 125I in the exposed portion of each forelimb. For each tumor an uptake ratio (UR) was calculated for the radioactivity in the tumor compared to muscle. With this shield apparatus, reproducible, sensitive and relatively rapid determinations of UR values can be obtained. Antibody localization can be detected in some tumors which are nonpalpable or very small and for tumors of up to 2 cm3 in volume. Whole-body radioactivity ranging from less than 0.037 MBq to higher levels suitable for radioimmunoscintigraphy could be utilized in this procedure.

Improved tumor localization of 111In labeled monoclonal antibody with chelator administration to host nude mice.

Administration of calcium disodium edetate (EDTA) substantially increased the tumor:muscle ratios of nude mice injected with 111In labeled monoclonal antibody 9.2.27. This effect was apparent 3 days to 7 days after antibody injection of mice bearing the BRO human melanoma, but not earlier. The tumor:muscle ratios decreased during this time for animals not receiving chelator. Contemporaneously, EDTA treated mice lost whole body radioactivity more rapidly than did their untreated counterparts, suggesting that 111In which had dissociated from the antibody-DTPA-radiometal complex was chelated and excreted. These results suggest that effective chelator treatment might improve tumor localization of radioactivity after injection of 111In labeled antibodies.

In vivo cytotoxicity assays with 111In-labeled tumor cells: comparison with [125I]iododeoxyuridine-labeled cells and effects of chelators and inoculation site.

The fate of cells inoculated in nude mice can be determined using human tumor cells prelabeled with 111In oxine (111InOx). To facilitate elimination of extracellular 111In released from killed cells, an appropriate metal chelating agent is administered to the host mice. With calcium disodium edetate administered by various routes, highly significant differences in the rates of 111In loss were observed for viable compared to killed cells after i.p. or i.m. inoculation. Radioactivity was eliminated at similar rates for mice bearing viable cells labeled with 111In or with [125I]iododeoxyuridine (125IUdR). For killed cells, 111In was eliminated somewhat less efficiently than 125I. The 111In-prelabeling method allows facile and sensitive determination of host-mediated cytotoxicity for inoculated tumor cells.

Quantitative evaluation of anticancer agents against human melanoma cells implanted in nude mice.

BRO human melanoma cells, which are exceptionally tumorigenic and lethal for nude mice, were inoculated intraperitoneally or intracerebrally in varying numbers. An inverse linear correlation was observed between the logarithm of the number of cells inoculated and host survival. In mice bearing 10(7) cells intraperitoneally, 2.4-2.8 log10 units of cell kill were obtained after a single intraperitoneal injection of vincristine, and some mice inoculated with 10(5) cells were cured by this treatment. Fewer cells were killed by L-phenylalanine mustard. Vincristine did not prolong survival of nude mice with intracerebral BRO tumors. Cell kill after administration of anticancer agents can be quantitated for BRO cells inoculated intraperitoneally or intracerebrally.

Prediction of anticancer activity by tumor uptake of radiolabeled monoclonal antibody.

A novel and sensitive method is described for rapid detection of anticancer drug efficacy against human tumor xenografts in nude mice. CA or BRO human melanoma cells were inoculated i.m. in nude mice, which then were treated with saline or cytotoxic agents. After injection of 111In-labeled 9.2.27 antimelanoma monoclonal antibody, the site of tumor inoculation and the contralateral non-tumor site were excised from sacrificed mice. Relatively lower 111In uptake in the area of tumor inoculation correlated with prevention or delay of tumor growth in identically treated counterpart mice.

Selective cytotoxicity of 5-hydroxyuridine for human colon adenocarcinoma cells.

The cytotoxicity of 5-hydroxyuridine (OHUrd), 5-FU, and 5-fluorodeoxyuridine was determined by colony assays for three human colon adenocarcinoma cell lines and for a cell line derived from normal fetal intestinal cells. All three tumor cell lines were more sensitive to OHUrd than were the FeInt cells, whereas 5-FU was more toxic to the latter. 5-Fluorodeoxyuridine was substantially more cytotoxic to only one of the tumor cell lines compared to the normal cells. In HT-29 tumor cells, OHUrd cytotoxicity could be prevented by coincubation with uridine or cytidine but not by pyrimidine deoxyribonucleosides or by purine nucleosides. Compared to OHUrd, 5-hydroxyuracil was much less cytotoxic for HT-29 cells. The increased cytotoxicity of OHUrd for these tumor cells compared to fetal intestinal cells may implicate biochemical differences that might be exploitable for improved chemotherapy. Direct comparison of drug sensitivity of colon tumor cells compared to normal counterpart cells may provide a method of screening agents selective for these tumor cells.

Effectiveness of anticancer drugs determined in nude mice inoculated with [125I]5-iodo-2'-deoxyuridine-prelabeled human melanoma cells.

Anticancer drugs were tested on NIH-2 nude mice inoculated ip with BRO human melanoma cells, which are rapidly lethal for these hosts. Criteria for drug activity were a) increased host survival and b) an increased rate of radioactivity loss from mice bearing BRO cells prelabeled with [125I]5-iodo-2'-deoxyuridine. Diphtheria toxin, which is selectively toxic to human cells compared to mouse cells, prolonged host survival and accelerated 125I elimination in a dose-dependent manner. Drugs that increased the rate of 125I loss compared to the rate of untreated mice also prolonged the lives of treated mice. With one exception, drugs that did not accelerate 125I elimination had little or no effect on the length of survival.

Antitumor activity and minimal toxicity of concentrated thymidine infused in nude mice.

To avoid infusing large volumes of fluid while treating patients with the standard thymidine solution (30 g/liter), it may be possible to administer this drug in more concentrated form. At 25 degrees C, thymidine is saturating at a concentration of 52 g/liter of 0.6% NaCl solution, and the thymidine concentration at saturation increases with temperature. Nude mice were infused at 29 degrees C with thymidine (60 or 72 g/liter) in cycles consisting of 4 to 5 days infusion followed by 9 days rest. Therapeutically effective doses of concentrated thymidine did not cause significant mortality in mice, and weight loss attributable to treatment was small and reversible. Significant growth inhibition of CA 1 human melanoma heterotransplants was observed after 3 treatment cycles. After 4 or 5 cycles, tumor responses were obtained in 7 mice (6 complete responses) of 12 inoculated with this tumor. These results show that concentrated thymidine solutions are highly effective against human tumor heterotransplants in nude mice and suggest that clinical use of concentrated thymidine may allow practical administration of maximum tolerated doses of this drug.

Exceptional lethality for nude mice of cells derived from a primary human melanoma.

BRO human melanoma cells, obtained from a biopsy of a highly aggressive and malignant primary tumor, were grown as xenografts in nude mice and in cell culture. These cells were exceptionally tumorigenic and malignant for nude mice. NIH-II nude mice survived 11.0 +/- 0.4 (S.E.) and 14.1 +/- 0.4 days after i.p. inoculation of 10(7) or 10(6) BRO cells, respectively, and lethal tumors developed in all mice inoculated i.p. with only 10(3) cells. The doubling time (2.3 days) of the volume of tumors formed after s.c. inoculation was comparable to the doubling time of these cells in culture. After i.p. or s.c. inoculation, BRO cells metastasized to the diaphragm and lungs, causing respiratory failure in most of the host mice. The original tumor and the cell line derived from it had undifferentiated structures with prominent nuclei and very large nucleoli. Karyotype abnormalities included a gigantic A group chromosome, a large D group chromosome, and an unusual double centromere chromosome not found typically in human melanoma cells. Due to the short and reproducible survival times of nude mice inoculated i.p. with BRO cells, this model system may be useful for rapidly determining the effects of experimental treatment on the survival of hosts bearing human tumor cells.

111Indium labeling of cultured human tumor cells.

At least one microCi of 111indium oxine (111InOx) could be incorporated into 10(6) human tumor cells without cytotoxicity as determined by colony assay or by trypan blue staining. 111In bound to prekilled cells was removed preferentially by washings, and prelabeled cells killed with diphtheria toxin released radioactivity much more rapidly than did viable cells. The metal chelator calcium disodium edetate did not facilitate 111In removal from either viable or dead cells. High sensitivity can be obtained for in vitro or in vivo cytotoxicity assays using human cells prelabeled with 111InOx.

Spectroscopic studies of ternary complexes of thymidylate synthetase, deoxyribonucleotides, and folate analogs.

Conformational changes accompanying the formation of binary and tightly bound ternary complexes of thymidylate synthetase and all possible combinations of three folate analogs (N-10-ethyl-quinazoline, folic acid triglutamate, and folic acid) and three deoxyribonucleotides (5-fluoro-2'-deoxyuridylic acid (FdUMP), 2'-deoxyuridylic acid (dUMP), and thymidylic acid (dTMP] were studied by means of ultraviolet difference spectroscopy. The amplitudes of the spectral changes upon ternary complex formation were 2-3-fold greater than those generated by formation of binary enzyme-nucleotide and enzyme-folate analog complexes. Difference spectra of the ternary complexes all showed a major increase in absorbance in the region of 320-340 nm, presumably due to perturbations of the folate analog chromophores, whereas decreases in absorbance occurred over a range of 260-310 nm. N-10-ethyl-quinazoline tended to form the complex with the greatest filtration efficiency on nitrocellulose filters, followed by folic acid triglutamate and folic acid, whereas among the nucleotides, the most stable complexes were formed with FdUMP, followed by dUMP and dTMP. A correlation was observed between the apparent stability of the ternary complex and the magnitude of the absorbance change in its difference spectrum. The formation of the various ternary complexes showed three different categories of rate behavior: 1) very rapid formation of the complex; 2) biphasic formation with a rapid phase and a slow phase requiring up to 90 min for completion; and 3) in the case of the ternary complex formed with enzyme, FdUMP, and folic acid, only a slow phase of binding. The slow formation of the latter complex was accompanied by concomitantly slow changes in the difference spectrum. However, in those cases of biphasic formation of the complexes, almost all of the spectral change occurred rapidly, and very little of it corresponded to the slow phase of complex formation. To accommodate these observations, a model is proposed involving a sequential interaction of the two subunits of thymidylate synthetase.

Determination of cytotoxicity in vivo using 111indium-labeled human tumor cells.

Loss of radioactivity from nude mice was determined after inoculation of human tumor cells prelabeled with [111In]indium oxine (111InOx). Elimination of 111In was increased somewhat by treating the mice with diphtheria toxin (DT), which is toxic selectively for human cells compared to mice. Calcium disodium edetate (CaNa2EDTA), a metal chelating agent, facilitated elimination of 111In and increased the difference in the rates of loss of radioactivity from mice bearing viable compared to DT-killed cells.

Correlation of cytotoxicity with elimination of iodine-125 from nude mice inoculated with prelabeled human melanoma cells.

BRO human melanoma cells were prelabeled in vitro with [125I]5-iodo-2'-deoxyuridine ([125I]IdUrd) and inoculated into NIH-II nude mice ip, im, sc, or iv. Saline or diphtheria toxin (DT), which is selectively toxic to human cells compared to those of mice, was injected, and the loss of 125I from the animals was monitored daily with a whole-body gamma scintillation detector. For most of the inoculation sites DT accelerated the rate of 125I excretion and in all cases was cytotoxic for the inoculated cells as determined by host survival or measurement of visible tumor growth. Differences between the rates of 125I loss for DT-treated mice compared to untreated mice were most evident for cells inoculated ip or im. These results indicate that [125I]IdUrd prelabeling of human tumor cells inoculated in nude mice offers a rapid method for determination of cytotoxicity in vivo.

Cytotoxic and biochemical effects of thymidine and 3-deazauridine on human tumor cells.

Cytotoxicity and perturbations of the deoxyribonucleoside triphosphate pools caused by thymidine were studied in thymidine-sensitive and -resistant human tumor cells. Incubation with 1 mM thymidine reduced cell viability by more than 90% in the three sensitive cell lines (two melanomas and one adrenal carcinoma) and reduced the growth rate without decreasing the viability of resistant LO melanoma cells. Thymidine (1 mM) greatly increased the ratio of the deoxythymidine 5'-triphosphate to deoxycytidine 5'-triphosphate pools in the sensitive cells compared to LO cells and also caused larger relative increases in the pool sizes of deoxyguanosine 5'-triphosphate and deoxyadenosine 5'-triphosphate in the sensitive compared to the resistant cells. 3-Deazauridine, known to inhibit synthesis of deoxycytidine 5'-triphosphate and cytidine 5'-triphosphate in other cell lines, potentiated the cytotoxicity of thymidine for thymidine-sensitive BE melanoma and LO cells. In LO cells, 3-deazauridine (50 microM) decreased the intracellular pool of deoxycytidine 5'-triphosphate to the level obtained with 1 mM thymidine. Lower concentrations of deoxycytidine as compared to cytidine were required to protect BE and LO cells against the cytotoxicity of thymidine plus 3-deazauridine. Deoxycytidine also was more effective than was cytidine in preventing loss of cell viability after exposure to thymidine or to 3-deazauridine individually. In these human melanoma cells, ribonucleotide reductase may be a major site of action of thymidine, of 3-deazauridine, and of both drugs in combination. These results indicate that in human tumor cells the cytotoxic effect of thymidine correlates with greater perturbations of the pyrimidine deoxyribonucleoside 5'-triphosphate pools and that thymidine and 3-deazauridine, which independently reduce the intracellular levels of deoxycytidine 5'-triphosphate, act synergistically against human tumor cells.