Chemoresistance induces enhanced adhesion and transendothelial penetration of neuroblastoma cells by down-regulating NCAM surface expression

Abstract

Background: Drug resistance to chemotherapy is often associated with increased malignancy in neuroblastoma (NB). One explanation for the link between resistance and malignancy might be that resistance facilitates cancer progression and invasion. To investigate this hypothesis, adhesion, transendothelial penetration and NCAM (CD56) adhesion receptor expression of drug-resistant versus drug-sensitive NB tumor cells were evaluated.

Methods: Acquired drug resistance was mimicked by exposing parental UKF-NB-2, UKF-NB-3 or IMR-32 tumor cells to increasing concentrations of vincristine- (VCR) or doxorubicin (DOX) to establish the resistant tumor cell sublines UKF-NB-2VCR, UKF-NB-2DOX, UKF-NB-3VCR, UKF-NB-3DOX, IMR-32VCR and IMR-32DOX. Additionally, the malignant behaviour of UKF-NB-4, which already possessed the intrinsic multidrug resistance (MDR) phenotype, was analyzed. UKF-NB-4 exposed to VCR or DOX were designated UKF-NB-4VCR or UKF-NB-4DOX. Combined phase contrast - reflection interference contrast microscopy was used to separately evaluate NB cell adhesion and penetration. NCAM was analyzed by flow cytometry, western blot and RT-PCR.

Results: VCR and DOX resistant tumor sublines showed enhanced adhesion and penetration capacity, compared to their drug naïve controls. Strongest effects were seen with UKF-NB-2VCR, UKF-NB-3VCR and IMR-32DOX. DOX or VCR treatment also evoked increased invasive behaviour of UKF-NB-4. The process of accelerated tumor invasion was accompanied by decreased NCAM surface and protein expression, and down-regulation of NCAM coding mRNA. Transfection of UKF-NB-4VCR cells with NCAM cDNA led to a significant receptor up-regulation, paralleled by diminished adhesion to an endothelial cell monolayer.

Conclusion: It is concluded that NB cells resistant to anticancer drugs acquire increased invasive capacity relative to non-resistant parental cells, and that enhanced invasion is caused by strong down-regulation of NCAM adhesion receptors.

Figure 1

Adhesion kinetics of parental control cells (UKF-NB-2, UKF-NB-3, UKF-NB-4, IMR-32) versus vincristine (UKF-NB-2^VCR, UKF-NB-3^VCR, UKF-NB-4^VCR, IMR-32^VCR) or doxorubicin resistant subpopulations (UKF-NB-2^DOX, UKF-NB-3^DOX, UKF-NB-4^DOX, IMR-32^DOX). Adhesion capacity is depicted as tumor cell adhesion/mm² (mean ± SD; n = 6). X-axis indicates the period of co-culture. *indicates significant difference to the controls.

Figure 2

Penetration kinetics of parental control cells (UKF-NB-2, UKF-NB-4) versus vincristine (UKF-NB-2^VCR, UKF-NB-4^VCR) or doxorubicin resistant subpopulations (UKF-NB-2^DOX, UKF-NB-4^DOX). To identify transmigrated NB from the cells which had bound to HUVEC, a reflection interference contrast microscope with a Ploem apparatus was used (see materials and methods for details). X-axis indicates the period of co-culture. *indicates significant difference to the controls.

Figure 3

NCAM surface expression on UKF-NB-2, UKF-NB-3, UKF-NB-4, IMR-32 controls and the respective doxorubicin- (DOX) or vincristine resistant sublines (VCR). An FITC-conjugated monoclonal antibody anti- CD56, clone 16.2, was used to detect the NCAM 120, 140 and 180 kDa isoform. A mouse IgG1-FITC served as the isotype control (IgG). The figure shows the histogram analysis of one representative experiment. The complete experimental data set (MFU +/- SD; n = 6) is given above the histograms.

Figure 4

Confocal analysis of NCAM distribution. UKF-NB-4 control versus UKF-NB-4^VCR tumor cells were grown in standard medium. FITC conjugated monoclonal antibody clone 16.2 was used to analyze NCAM. The figure shows distinct NCAM expression at the intercellular boundaries on UKF-NB-4 control cells which is reduced in UKF-NB-4^VCR. Scale = 10 μM. × 100/1.3 oil immersion objective.

Figure 5

Western blot analysis of NCAM from the proteins of UKF-NB-3, UKF-NB-4, IMR-32 (Ctrl) versus vincristine (VCR) or doxorubicin resistant subpopulations (DOX). Cell lysates were incubated with anti NCAM (clone 16.2) monoclonal antibodies, which detect the NCAM 120, 140 and 180 kDa isoform. beta-actin served as the internal control. The figure shows one representative from three separate experiments.

Figure 6

Semi quantitative RT-PCR analysis of NCAM 140 kDa and 180 kDa RNA in parental UKF-NB-2, UKF-NB-3, UKF-NB-4, IMR-32 (Ctrl) versus vincristine (VCR) or doxorubicin resistant subpopulations (DOX). RNA were extracted, reverse-transcribed, and submitted to semiquantitative reverse transcription-PCR using gene specific primers as indicated in materials and methods. The internal control for the RT-PCR reaction was performed by running parallel reaction mixtures with the housekeeping gene GAPDH. The figure shows one representative from three separate experiments.

Figure 7

Adhesion kinetics of UKF-NB-4^VCR and of UKF-NB-4^VCR transfected with 2 μg of full-length cDNA encoding the human NCAM-140 kDa isoforms are shown. Control cells were transfected with the expression vector alone. Data on NCAM surface expression, evaluated by flow cytometry, are given in the lower right hand corner. 100% value = 51.4 ± 18.7 MFU. X-axis indicates the period of co-culture (mean ± SD, n=3). *indicates significant difference to UKF-NB-4^VCR.