Anti-LRP/LR-specific antibody IgG1-iS18 impedes adhesion and invasion of pancreatic cancer and neuroblastoma cells

Abstract

Background: Cancer has become a global burden due to its high incidence and mortality rates, with an estimated 14.1 million cancer cases reported worldwide in 2012 particularly as a result of metastasis. Metastasis involves two crucial steps: adhesion and invasion, and the non-integrin receptor; the 37-kDa/67-kDa laminin receptor precursor/ high affinity laminin receptor (LRP/LR) has been shown to be overexpressed on the surface of tumorigenic cells, thus being implicated in the enhancement of these two crucial steps. The current study investigated the role of LRP/LR on the aggressiveness of pancreatic cancer (AsPC-1) and neuroblastoma (IMR-32) cells with respect to their adhesive and invasive potential.

Methods: AsPC-1 and IMR-32 cells were utilized as the experimental cell lines for the study. Cell surface LRP/LR levels were visualised and quantified on the experimental and control (MCF-7) cell lines via confocal microscopy and flow cytometry, respectively. Total LRP/LR levels in the cell lines were assessed by Western blotting and the adhesive and invasive potential of the above-mentioned cell lines was determined before and after supplementation with the anti-LRP/LR specific antibody IgG1-iS18. Statistical significance of the data was confirmed via the use of the two-tailed student's t-test and Pearson's correlation coefficient.

Results: Flow cytometry revealed that AsPC-1 and IMR-32 cells displayed significantly higher cell surface LRP/LR levels in comparison to the MCF-7 control cell line. However, Western blotting and subsequent densitometric analysis revealed that all three tumorigenic cell lines displayed no significant difference in total LRP/LR levels. The treatment of AsPC-1 and IMR-32 cells with IgG1-iS18 caused a significant reduction in the adhesive and invasive potential of the cells to laminin-1 and through the ECM-like Matrigel™, respectively. Pearson's correlation coefficients indicated a high correlation, thus suggesting a directly proportional relationship between cell surface LRP/LR levels and the adhesive and invasive potential of AsPC-1 and IMR-32 cells.

Conclusion: These findings suggest that through the interference of the LRP/LR-laminin-1 interaction, the anti-LRP/LR specific antibody IgG1-iS18 may act as an alternative therapeutic tool for the treatment of metastatic pancreatic cancer and neuroblastoma.

Keywords: 37 kDa/67-kDa laminin receptor (LRP/LR); Adhesion; Invasion; Laminin-1; Metastasis; Neuroblastoma; Pancreatic cancer.

Fig. 1

Visualisation of cell surface LRP/LR levels of pancreatic cancer (AsPC-1) and neuroblastoma (IMR-32) cells. The cells were non-permeabilized in order to visualize the surface of the cancerous cells. a The cells were labelled with the primary antibody IgG1-iS18 which binds to LRP/LR and the FITC-coupled secondary antibody which is specific for the primary antibody thus exhibiting green fluorescence. b The cells were labelled with the anti-chloramphenicol acetyltransferase (CAT) antibody as the negative control. c The cells were labelled with the FITC coupled secondary antibody only, to indicate that no non-specific binding of the secondary antibody occurred

Fig. 2

Detection of LRP/LR levels on the surface of pancreatic cancer and neuroblastoma cells. a Quantification of LRP/LR on the surface of pancreatic cancer (AsPC-1) and neuroblastoma (IMR-32) cells. The black peak represents the unlabelled cells, whilst the blue peak represents the cells labelled with goat anti-human phycoerythin (PE)-coupled secondary antibody. The red peak represents the cells labelled with both anti-LRP/LR specific antibody IgG1-iS18 and the afore-mentioned secondary antibody. The inclusion of the unlabelled cells as a control, confirms that the secondary antibody does not bind non-specifically. The shift observed between the black and red peak indicates a change in fluorescence intensity thus the presence of LRP/LR on the cell surface of the cell lines under study. b Determination of chloramphenicol acetyltransferase protein levels on the surface of the three tumorigenic cell lines as a control. The blue peak represents cells that have been labelled with both anti-CAT primary antibody and goat anti-rabbit allophycocyanin (APC)-coupled secondary antibody, whilst the red peak represents the cells labelled with the afore-mentioned secondary antibody only. No distinct shift is seen between the blue and red peak, thereby indicating the absence of the CAT protein on the surface of the cell lines under study

Fig. 3

Quantification of cell surface LRP/LR levels on pancreatic cancer (AsPC-1) and neuroblastoma (IMR-32) cells. The cells were labelled with the primary IgG1-iS18 antibody and the anti-human phycoerythrin (PE) secondary antibody. 20 000 cells were analysed across all three cell lines, with the median fluorescence intensity analysed as an indicator of the cell surface LRP/LR levels. In comparison to the MCF-7 control cell line, AsPC-1 cells exhibit an approximate 2-fold increase in cell surface LRP/LR whilst the IMR-32 cells exhibit an approximate 4-fold increase in cell surface LRP/LR. This data is representative of three experimental triplicates. The MCF-7 values were set to 100%. The error bars represent standard deviation and p-values *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001

Fig. 4

Detection of total LRP levels in pancreatic cancer (AsPC-1) and neuroblastoma (IMR-32) cells. a Western blot analysis was performed to detect total 37-kDa LRP levels in all three cancer cell lines. β-actin was used as a loading control. b Densitometric quantification performed on these blots revealed that there was no significant difference in total LRP/LR levels between the poorly invasive MCF-7 cells and the two experimental cell lines: AsPC-1 and IMR-32. This data is representative of an average of three experiments. The values obtained from quantification of LRP were divided by the values obtained from β-actin quantification and the resultant values were used to construct the above graph. The MCF-7 values were set to 100%. The error bars represent standard deviation and Non-significant (N.S) p-value: p > 0.05

Fig. 5

IgG1-iS18 reduces the adhesive potential of AsPC-1 and IMR-32 cells to laminin-1. Absorbance was measured at 550 nm after an incubation of one hour and after staining with crystal violet. CAT was used as a negative control. When treated with IgG1-iS18, the adhesive potential of the AsPC-1 and IMR-32 cells decreased significantly. The MCF-7 untreated value was set to 100%. The error bars represent standard deviation and p-values *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, Non-significant (N.S): p > 0.05

Fig. 6

IgG1-iS18 reduces the invasive potential of AsPC-1 and IMR-32 cells through the ECM-like Matrigel™. Absorbance was measured at 620 nm after an incubation of 24 h and after staining with toluidine blue. CAT was used as a negative control. When treated with IgG1-iS18, the invasive potential of the AsPC-1 and IMR-32 cells was significantly decreased, respectively. The MCF-7 untreated value was set to 100%. The error bars represent standard deviation and p-values *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, Non-significant (N.S): p > 0.05