Materials and methods

Cell culture

Human nasopharyngeal squamous cell carcinoma cells, FaDu, were obtained from the American Type Culture Collection (Manassa, VA), and maintained in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Paisley, UK) supplemented with 10% fetal calf serum (Sigma, Poole, UK) and 2 mM L-glutamine (Life Technologies). Cells were kept in a humidified incubator at 37°C and 5% CO₂/air. Cells were routinely subcultured in 75 cm² cantilevered flasks.

Cells were transfected with either the HRP gene (pssHRP-puro), or the marker green fluorescent protein (GFP, pEGFP-puro) using a cationic lipid and integrin-targeted peptides, as previously described.^{2, 9} In both plasmids, transgene expression was under the control of the strong cytomegalovirus (CMV) promoter. Cells were selected in puromycin containing media (1 g/ml, Sigma) and colonies isolated and expanded. Gene expression was confirmed by fluorescent-activated cell sorting (FACS) for GFP, or enzyme activity, using a modified 3,3',5,5'-tetramethylbenzidine assay (TMB),² for HRP. HRP activity was detected in HRP and not GFP transfectants (results not shown). Single clones were isolated, named HRP8 and GFP1, and stable cell lines derived from these initial clones were used throughout the experiments. Cells were confirmed as mycoplasma negative using a PCR method (ATCC Mycoplasma Detection Kit Version 2.0).

Monolayer clonogenic assays

Exponentially growing cells were collected from monolayer culture by trypsinization and plated at low density. Cells were allowed to adhere for 4–6 hours. Prodrugs were dissolved in Hanks' balanced salt solution (HBSS, Life Technologies) and cells exposed in the 37°C incubator.

Following drug exposure, cells were washed in phosphate-buffered saline (PBS, Oxioid Ltd, Basingstoke, UK) and grown for approximately 10 days in complete media supplemented with feeder cells (heavily irradiated V79 cells). Colonies were fixed in 0.25% methylene blue w/v in 50% isomethylated spirit (IMS). Colonies estimated to be greater than 50 cells were counted, and survival calculated relative to vehicle-treated controls.

Spheroid formation

Exponentially growing cells were trypsinized and plated at 10⁵/ml in CO₂ Independent Media (Life Technologies) supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin (Life Technologies), with 3 ml of cells per well of a six-well plate. Plates were incubated in a 37°C warm room on a rotating platform (R100, Rotatest rotator) set at 75 rpm to prevent cell adhesion. The medium was replaced on the third day, and subsequently every 2 days. After 3 days, visible spheroids had formed, and were measured using a calibrated eyepiece graticule (Graticules Ltd, Kent, UK).

Immunohistochemistry of spheroids

Spheroids of varying sizes were exposed to 100 M Pimonidazole (Hypoxyprobe-1, Natural Pharmacia Int Inc.) for 2 hours to stain hypoxic cells.¹⁰ Spheroids were washed in PBS, and frozen in embedding compound (Sakura). Sections (5 m) were cut, air dryied and fixed in ice-cold acetone. Sections were stored at -20°C prior to antibody staining. Spheroids were also incubated under catalyst-induced anoxia (anoxic glove cabinet, Don Whitley Scientific, UK) for 2 hours prior to the addition of Pimonidazole. Spheroids were processed as those treated under normoxia.

Pimonidazole staining was carried out using an anti-pimonidazole IgG1 antibody (Natural Pharmacia) diluted 1:100 in Tris-buffered saline with protein block (Dako, UK) and an UltraVision Mouse Tissue Detection System (Stratech Scientific, UK), following the manufacturer's instructions. Staining was identified using the diaminobenzidine (DAB) chromophore, and slides were counterstained with hematoxylin.

Spheroid clonogenic assay

Intact spheroids were exposed to prodrugs dissolved in HBSS on the rotating platform for 4 or 24 h. Following exposure, whole spheroids were collected, washed, trypsinized and syringed to give a single cell suspension, counted, and appropriate numbers of viable cells plated on 6 cm dishes. Feeder cell supplemented media was added and colonies allowed to form as described above.

Statistical analysis

JMP statistical analysis program was used to carry out ANOVA and t-test analyses.

Results

The FaDu tumor cells readily formed spheroids when grown on a continuously rotating platform. Spheroids grew uniformly, and reached 300 m diameter after approximately 3 days in culture, and 700 m after 7–10 days. When frozen sections were stained with hematoxylin and eosin, large spheroids showed regions of loosely packed necrotic cells in the center of the spheroid. In addition, Pimonidazole (a bioreductively activated hypoxia marker) treatment of spheroids showed an increase in staining, and hence concomitant increase in hypoxia, towards the center of the spheroid (Fig 1a). Spheroids incubated under anoxia and then treated with Pimonidazole showed staining throughout the entire spheroid (Fig 1b).

Figure 1.

(a) Spheroid of 600 m diameter stained with Pimonidazole to show regions of hypoxia (in brown). Staining starts approximately 150 m from the surface, and increases in intensity towards the center with a loosely packed necrotic center. (b) pimonidazole staining in 600 m diameter spheroid maintained under anoxic conditions for 2 hours prior to and during Pimonidazole incubation.

Full figure and legend (452K)

Exposure of monolayers to IAA for 4 hours (Fig 2a) or 24 hours (Fig 3a) resulted in a decrease in the clonogenicity of HRP transfectants compared with GFP controls, with over 2-log of kill following 24-hour exposure to high IAA concentrations. The surviving fraction for 24-hour exposure showed a slight plateau at 0.5 mM IAA, with survival decreasing steeply at concentrations above 1 mM, similar to the plateau seen previously in transient transfectants.² 4 h exposure of 300 m diameter spheroids to IAA (Fig 2b) resulted in a steep decline in survival at concentrations greater than 1 mM, with a greater decrease than in monolayers. Survival of cells from 700 m diameter spheroids (Fig 2c) was similar to cells exposed as monolayers. After 24 hours exposure to IAA, both 300 and 700 m diameter spheroids showed similar survival curves to monolayers. However, the plateau phase in the larger spheroids was more pronounced than in monolayers or in smaller spheroids (Fig 3).

Figure 2.

Clonogenic survival following 4-hour exposure to IAA of (a) monolayers, (b) 300 m diameter and (c) 700 m diameter spheroids. Closed circles, GFP1 controls; crosses, HRP8 cells. Data are meanSEM of three independent experiments, triplicate samples.

Full figure and legend (23K)

Figure 3.

Clonogenic survival following 24-hour exposure to IAA of (a) monolayers, (b) 300 m diameter and (c) 700 m diameter spheroids. Closed circles, GFP1 controls; crosses, HRP8 cells. Data are meanSEM of three independent experiments, triplicate samples.

Full figure and legend (23K)

The halogenated-IAA derivative, 5Br-IAA, was also evaluated as a prodrug in this system. In monolayers exposed to 5Br-IAA for 4 hours, there was no effect on the survival of either GFP1 or HRP8 cells (Fig 4a). However, after 24 hours exposure clonogenicity was markedly reduced with over 2 log of cell kill at just 1 mM in HRP8 cells (Fig 5a). In the small 300 m diameter spheroids, which lack a necrotic center, 4 hour 5Br-IAA exposure again had very little effect (Fig 4b). However, in the 700 m diameter spheroids, HRP8 cell survival was reduced by 50% at just 0.1 mM (Fig 4c).

Figure 4.

Clonogenic survival following 4-hour exposure to 5Br-IAA of (a) monolayers, (b) 300 m diameter and (c) 700 m diameter spheroids. Closed circles, GFP1 controls; crosses, HRP8 cells. Data are meanSEM of three independent experiments, triplicate samples.

Full figure and legend (26K)

Figure 5.

Clonogenic survival following 24-hour exposure to 5Br-IAA of (a) monolayers, (b) 300 m diameter and (c) 700 m diameter spheroids. Closed circles, GFP1 controls; crosses, HRP8 cells. Data are meanSEM of three independent experiments, triplicate samples.

Full figure and legend (25K)

In both 300 and 700 m diameter spheroids, 24 hour exposure to the halogenated indole resulted in approximately 1 log cell kill in HRP8 cells (Figs 5b and c), which was markedly less than that seen in monolayers (Fig 5a). However, a clear increase in cell debris with a concurrent loss of intact spheroids in the treated spheroid cultures was evident, which was not seen in untreated, or control GFP cultures (Fig 6).

Figure 6.

Large, 700 m diameter spheroids exposed to IAA for 4 hours (a) GFP1 cells, (b) HRP8 cells. Original magnification 40.

Full figure and legend (164K)

Discussion

The growth of cells on a rotating platform prevents adhesion to the culture flask surface, resulting in the formation of multicellular spheroids, which subsequently grow in three dimensions. FaDu spheroids retained expression of transgenes, and were amenable to treatment with indole prodrugs. Consistent with reports of spheroids derived from a variety of other cell lines,^{10, 11, 12} FaDu spheroids developed a hypoxic center and subsequently a necrotic core when they reached approximately 500–600 m. This has been reported to be accompanied by alterations in the cell cycle distribution of cells, increases in the levels of carbon dioxide at the core, and also the concentration of other waste products, such as lactic acid. This makes spheroids a more accurate model for the in vivo tumor situation, and in particular of avascular micrometastases.¹³

Both IAA and 5Br-IAA were effective in decreasing the clonogenic ability of HRP-transfected (HRP8) cells after prolonged exposure periods despite FaDu cells carrying a p53 missense mutation.^{8, 14} The concentrations of 5Br-IAA required to decrease clonogenicity were markedly (five-fold) lower than IAA concentrations which may make 5Br-IAA a more attractive prodrug in vivo.

However, the halogenated derivative appeared to be ineffective following short exposure of monolayers under normoxia, which is consistent with our previous reports in transient transfectants.⁵ The reason is currently unknown, but may be related to the drug activation pathway.

In multicellular spheroids, IAA resulted in similar levels of cell kill in both small and large HRP8 spheroids, indicating that the cytotoxicity of the final compound is unaffected by the range of conditions present in large spheroids. It may be that in the large spheroids, only the outer proliferating cells are metabolizing the conversion of IAA to the cytotoxin. In that case, the level of cell kill indicates that the active metabolite is capable of diffusing through the spheroid and killing quiescent cells. This is consistent with our reports of media transfer experiments showing that the bystander compound is freely diffusible.²

The 5Br-IAA compound has a different profile of toxicity when comparing monolayers with spheroids. We previously reported that in monolayers transiently expressing HRP, 5Br-IAA caused a marked decrease in cell kill after a 2 hour incubation period, but only when cells were exposed under anoxia.⁵ This suggested the involvement of a short-lived toxic radical species. In the current study, monolayers and small spheroids showed no decrease, whereas large 700 m diameter spheroids displayed a significant decrease in cell survival following 4 hour incubation. It is possible that this is due to the activation of 5Br-IAA in hypoxic regions, and subsequent bystander cell killing. It has been hypothesized that under oxic conditions methylene-2-oxindole (MOI) is involved in the cytotoxicity of IAA.³ However, under anoxia, MOI is not formed, and it is likely that an, as yet undetermined, toxic metabolite is involved. If 5Br-IAA is, as predicted, activated under hypoxia, then 4 hours incubation was sufficient for the drug to reach the hypoxic fraction within 700 m diameter spheroids. This indicates that the indole prodrugs would be able to reach cells distant from functional blood vessels.

The clonogenic assays are likely to underestimate the amount of cell kill in the spheroid model, since only whole spheroids are collected and only viable cells are plated. The loss of cells due to spheroid fragmentation was evident by microscopic examination but not quantifiable by the assay. For 300 m diameter spheroids exposed to IAA for 4 hours, the percentage of viable cells in HRP spheroids decreased to less than 10% at 5 mM supporting the theory of underestimation (data not shown).

Levels of cell kill in stable FaDu transfectants is lower than that previously reported for FaDu transient transfectants.^{5, 15} In addition, the plateau in cell kill seen at 1 mM concentrations of prodrug is more prominent in stable transfectants than in cells transiently expressing the HRP gene.² Levels of enzyme activity were similar however between the transfectant types. One possible reason for the discrepancies may be the enzyme activity per cell. Transient transfectants expressed HRP in approximately 10–14% of cells.¹⁵ Immunocytochemistry performed previously on stable transfectants showed low, uniform staining for HRP (results not shown), indicating that a far greater proportion of cells expressed the HRP enzyme, with the actual activity per cell being lower. Continuous expression of HRP is growth inhibiting to Escherichia coli.¹⁶ In the clinic, following in vivo gene delivery, a situation more closely related to transient transfectants will occur, with a mosaic of expressing and nonexpressing cells. In this case, cell kill may be greater than that seen in stable transfectants.

In conclusion, the HRP/IAA combination shows efficacy in an in vitro tumor model, which is promising for its application in vivo. In particular, the 5Br-IAA prodrug may be important in targeting hypoxia. However, further experiments are required to confirm whether the measured cell kill and potential cell kill are observable in animal growth delay models.

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Acknowledgements

Thanks to Dr. P Hoskin for discussions, and Dr K Sales and Dr A Brooks for spheroid suggestions. This work was funded by Cancer Research UK.