Prerequisites of an intracerebral in vivo model for the p-gp modu-

The orthotopic brain tumor model is suitable for studies on the p-gp modulation at the BBB (see also chapter 7). Furthermore, it is used for treatment experiments of primary and secondary brain tumors. Selected tumor cell lines for these tests have to comply with the following requirements. To observe a p-gp modulatory effect only at the BBB, the cells have to be negative for the MDR1 phenotype. As a second prerequisite, the cells should be chemosensitive in the presence of low concentrations of cytostatic drugs. In addition, the cells have to be tumorigenic in the brains of nude mice.

100 In vivo models of human lung cancer brain metastases 5.3.1.1 Investigations on the MDR phenotype of human lung cancer cell lines The MDR phenotype of the selected lung cancer cell lines NCI-H460 and DMS 114 was examined in three ways. The existence of p-gp encoding mRNA was detected by PCR.

The p-gp expression was demonstrated with immunohistochemical methods, and the func-tionality of an eventually expressed p-gp was determined by the calcein-AM efflux assay.

DNA-standard NCI-H460 DMS114

ß-actin 304 bp p-gp 167 bp 500 bp

300 bp 200 bp

Figure 5.1: Determination of p-gp encoding mRNA in NCI-H460 and DMS 114 cells. mRNA encoding for the MDR1 phenotype was only detected in NCI-H460 cells (ethidium-bromide stained RT-PCR gel after 34 cycles. β-Actin served as a control.)

The results of the RT-PCR for the MDR1 encoding mRNA are shown in Fig. 5.1.

mRNA encoding for p-gp was detected in PCR products of the NCI-H460 cells. DMS 114 cells possessed no p-gp encoding gene, because no PCR product of 167 base pairs was observed. β-Actin served as a control for proper RT-PCR procedure.

The expression of p-gp was examined with an avidin-biotin-method on paraffine sec-tions as well as on fixed cells. No typical p-gp staining was observed on any examined slide. Hence, although NCI-H460 cells have MDR1 encoding mRNA, p-gp is not expressed at the protein level. In case of the DMS 114 cell line, the negative MDR phenotype of the cells determined in the RT-PCR experiment was confirmed.

Fig. 5.2 shows the results of the calcein-AM efflux assay that was used to investigate the cell lines regarding p-gp activity. Both NCI-H460 and DMS 114 cells took up calcein-AM very well leading to high fluorescence intensities. Therefore, no active p-gp was expressed in the two cell lines despite the genetic disposition of H460 cells. However, NCI-H460 cells could presumably become MDR resistant by the addition of p-gp inducers like

5.3 Results 101

Figure 5.2: Comparison NCI-H460 (dashed line), DMS 114 (dotted line) and p-gp overexpress-ing Kb-V1/VBL (solid line) cells with respect to their p-gp activity. Both NCI-H460 and DMS 114 cells show higher fluorescence intensities compared to Kb-V1/VBL cells indicating an inactive p-gp or the lack of p-gp.

vinblastine.

In conclusion, the selected cell lines do not exhibit a MDR1 phenotype. Therefore, the study of p-gp modulation exclusively at the BBB is possible using NCI-H460 or DMS 114 cells in an orthotopic brain tumor model.

5.3.1.2 Chemosensitivity of human lung cancer cell lines

With respect to a successful treatment experiment the selected tumor cell lines have to be sensitive against chemotherapeutic agents. For our treatment studies the cytostatic drugs paclitaxel and vinblastine were chosen due to their applicability in treatment experiments in nude mice (Fellner et al. 2002, Spruss et al. 1995). Fig. 5.3 and Fig. 5.4 illustrate the chemosensitivity of DMS 114 and NCI-H460 cells against the two cytostatic drugs. Both substances are very potent cytostatics and thus, cytocidal effects were achieved in low concentrations. For example, 5 nM paclitaxel had a transiently cytocidal effect on DMS 114 cells, i.e. not all cells died and cells began to recover after 100 hours. In contrast, a concentration of 1 nM vinblastine was already sufficient for a permanent cytocidal effect.

In general, the NCI-H460 cells were less sensitive to the selected cytostatic drugs than the DMS 114 cells (see also chapter 3.3.4). Concentrations of 30 nM paclitaxel and 50 nM vinblastine, respectively, were necessary to achieve cytocidal effects.

102 In vivo models of human lung cancer brain metastases

Figure 5.3: Chemosensitivity of DMS 114 cells against paclitaxel (a) and vinblastine (b)

Figure 5.4: Chemosensitivity of NCI-H460 cells against paclitaxel (a) and vinblastine (b)

5.3 Results 103 Hence, NCI-H460 as well as DMS 114 cells were sensitive against low concentrations of the chemotherapeutic drugs paclitaxel and vinblastine.

5.3.1.3 Intracerebral in vivo growth

To investigate the tumorigenicity of the selected tumor cells three different tumor cell quantities were injected into the brain of nude mice to determine the optimal cell concen-tration. After three to four weeks, the intracerebral tumor growth was controlled. Fig.

5.5 and Fig. 5.6 illustrate an intracerebrally grown DMS 114 tumor. In this case 100,000 cells were injected.

The appearance of the brain tumor is similar to that of the subcutaneously grown tumor (see chapter 3.3.5). Here as well, the cells have mostly one prominent nucleolus.

Multinucleated cells are observed, too. The tumor is not well-delimited against the brain tissue. The infiltration into other brain areas is clearly visible.

In case of NCI-H460 tumors the characteristics of the subcutaneously grown tumor are similar to those of the intracerebral tumor (Fig. 5.7 and Fig. 5.8). The cells with scanty cytoplasm have round to oval nuclei with irregularly formed prominent nucleoli. Mitotic

50µm

Figure 5.5: MG-staining of an intracerebrally grown DMS 114 tumor. The diffuse tumor infil-trates other brain regions (indicated by arrows).

104 In vivo models of human lung cancer brain metastases

20µm

1

2

Figure 5.6: MG-staining of an intracerebrally grown DMS 114 tumor. The anisometrically formed cells are occasionally multinucleated (indicated by arrow 1). The irregular formed nuclei are of different size and have several prominent nucleoli (indicated by arrow 2).

100µm

Figure 5.7: MG-staining of an intracerebrally grown NCI-H460 tumor. Many blood vessels (indicated by arrows) are observed in this well-delimited tumor.

5.3 Results 105

20µm

1

2

3

Figure 5.8: MG-staining of an intracerebrally grown NCI-H460 tumor. The cells have round to oval nuclei with several irregularly shaped nucleoli. Mitotic activity (arrow 1) and the vascularization of the tissue (arrows 2 and 3) is marked by arrows.

activity is not as high as seen in the subcutaneously grown tumor. The tumor tissue is well vascularized, but infiltration of the vessels is not observed. After three weeks nearly all mice were dead because the tumor grew very rapidly and aggressively in the brain.

A tumor was detectable in all examined brains. Thus, both cell lines were tumorigenic in the nude mouse brain.

In summary, the selected lung cancer cell lines NCI-H460 and DMS 114 fulfill all three prerequisites. They are suitable to establish an orthotopic tumor model simulating brain metastases of lung cancer. Furthermore, the cell lines are sensitive to the selected chemotherapeutic agents. Since NCI-H460 and DMS 114 cells do not express a MDR1 phenotype, the effect of a specific p-gp modulation at the BBB could be examined in a treatment experiment.