Transferrin receptor content in tumor cells

8.3.1.1 TfR content of various tumor cell lines

The TfR content of several tumor cell lines that include brain and lung cancers was deter-mined in vitro by an indirect immunofluorescence detection procedure at a flow cytome-ter. The FITC fluorescence intensity was quantified in molecules of equivalent solubility (MESF) by means of four fluorescent standard microbead populations of approximately 7 to 9 µ in diameter. The beads matched both the excitation and emission spectra of FITC labelled antibodies. Each microbead population had a different level of fluores-cence intensity that referred to a certain MESF value. This means that microbeads with a MESF value of 10,000 exhibit the same fluorescence intensity as a solution containing 10,000 FITC molecules. A calibration curve was obtained by plotting the fluorescence intensities (relative channel number) of the single bead populations against the corre-sponding MESF values of the 4 populations. The single bead populations were recorded at the same settings as the analyzed samples (Fig. 8.3). Using this calibration curve, the MESF value of every sample was determined and measurements performed on different days could be compared with each other.

10⁰ 10¹ 10² 10³

Figure 8.3: (a) fluorescence intensity at FL1-H channel of a mixture consisting of 4 microbead populations. (b) calibration curve of MESF calculated from microbead fluorescence intensities. RCN (relative channel number) is equivalent to FL1-H channel number.

8.3 Results and discussion 183 The secondary antibody used for the indirect determination of TfR was labelled with FITC. Since the ratio of fluorescent dye and antibody ranges between 1 and 3, the fluo-rescence intensity of the different tumor cells can not be transformed directly in antibody binding capacity values or TfR densities. Direct quantification of receptor densities with fluorescent antibodies is only possible at fluorescent dye/antibody ratios of 1 as in case of phycoerythrin (PE) labelling. In consideration of the FITC/antibody ratio only a range for the TfR density of the different cell lines could be estimated. The TfR density ranges of selected glioblastoma and lung cancer cell lines are summarized in table 8.1. The TfR densities of the lung cancer cell lines were determined as these tumor entities metastasize preferentially to the brain. The human B-cell line RAJI served as a positive control for TfR expression.

Table 8.1: MESF values of different cell lines and their corresponding TfR density. Measure-ments were taken on day 5 after last passage (n= 3-6).

transferrin receptor cell line MESF [·10³] density [·10³]

min. max.

U-87 MG 347.91 ± 51.96 139 - 348 U-118 MG 714.07± 224.46 286 - 714 U-373 MG 540.36 ± 26.83 216 - 540

RAJI 158.50± 3.37 64 - 159

NCI-H460 266.06± 3.05 106 - 266

DMS 53 61.82 ± 1.21 25 - 62

DMS 114 75.86 ± 1.42 30 - 76

The highest TfR densities were detected on U-87 MG, U-118 MG and U-373 MG glioma cells with levels 2 to 4 times higher compared to those of the RAJI control cells. The examined lung cancer cell lines exhibited lower TfR densities than the brain tumor cells, particularly the small cell lung cancer cells DMS 53 and DMS 114. In these cell lines TfR densities under 100,000 were determined. In contrast, in NCI-H460 non-small cell lung cancer cells TfR is expressed at a level similar to that in U-87 MG cells. The TfR

184 The transferrin receptor - a possible loophole at the blood-brain-barrier density in the U-118 MG cells was the highest among the analyzed cell lines. However, the expression of TfR seemed to be unequal, since the standard deviation of the calculated MESF value was extremely high. Lower standard deviation combined with high TfR density was found in U-373 MG cells. Therefore, the U-373 MG cell line is the most suitable one among the examined brain tumor cell lines, for a TfR targeted treatment regarding the TfR density in vitro.

8.3.1.2 Growth depended TfR expression

The TfR is essential for cell growth as it mediates the transport of iron into the cell, which is necessary for metabolic processes. Several work groups identified a close relationship between TfR expression and cell proliferation (Trowbridge et al. 1984, Gambari et al. 1986, Chan et al. 1994). Therefore, we investigated the TfR content of various glioblastoma cell lines in different proliferation stages by indirect immunofluorescence detection using FITC labelled antibodies. RAJI cells are described as TfR expressing cells in literature (Laskey et al. 1988) and were used as a positive control. The human glioblastoma cell lines U-87 MG, U-118 MG and U-373 MG were examined 3, 5 and 7 days after passaging to simulate subconfluent and postconfluent growth. The measured fluorescence intensities of the single cell lines plotted against the growth time are depicted in Fig. 8.4.

In all cell lines a FITC fluorescence intensity that correlates with TfR expression was detected at all time points. For the control cell line RAJI (black bars) the highest flu-orescence intensities were measured after 3 days. Subsequently, the intensity strongly decreased to values 7 times lower than those measured after 3 days in culture. This indi-cated that the growth of the RAJI cells is almost complete after 3 days. Thus, there is no need for further TfR expression. In case of the U-87 MG cells (right-hatched bars) the determined fluorescence intensity reached maximum values on day 3 as well. Indeed, the intensities measured on the other two days slowly declined compared to day 3, but the decrease was not as distinct as observed in the RAJI cells. In contrast, the fluorescence in-tensity of U-118 MG cells (cross-hatched bars) increased up to day 5. After 2 further days of incubation only half of the fluorescence intensity was measured compared to day 5. The fluorescence intensity of U-373 MG cells (left-hatched bars) remained relatively constant over the examination period in consideration of the SEM values. The detected intensity

8.3 Results and discussion 185

Figure 8.4: Growth dependent TfR expression in different glioblastoma cell lines U-87 MG (right-hatched bars), U-118 MG (cross-hatched bars) and U-373 MG (left-hatched bars). RAJI cell line (black bars) serves as control for measurable TfR expression.

(mean values±SEM)

values were similar to those measured in U-87 MG cells, but U-373 MG cells showed no decrease in fluorescence intensity. This indicated a relatively constant cell growth in vitro.

In view of the suitability for the mentioned tumor model, the brain tumor cell line should have a constant TfR expression over a longer period of time. This requirement is best fulfilled by the U-373 MG cells as they showed the most constant TfR expression.