Using breast cancer cell CXCR4 surface expression to predict liposome binding and cytotoxicity

Abstract

The primary cause of mortality in breast cancer is tumor aggressiveness, characterized by metastases to regional lymph nodes, bone marrow, lung, and liver. C-X-C chemokine receptor type 4 (CXCR4) has been shown to mobilize breast cancer cells along chemokine gradients. Quantification of CXCR4 surface expression may predict the efficacy of anti-CXCR4 labeled liposomal therapeutics to target and kill breast cancer cells. We evaluated gene and surface receptor expression of CXCR4 on breast cancer cell lines distinguished as having low and high invasiveness, MDA-MB-175VII and HCC1500, respectively. CXCR4 surface expression did not correlate with invasiveness. MDA-MB-175VII exhibited more binding to anti-CXCR4 labeled liposomes relative to HCC1500. Increased binding correlated with greater cell death relative to IgG labeled liposomes. Quantitative cell characterization may be used to select targeted therapeutics with enhanced efficacy and minimal side effects.

Figure 1

Schematic illustration of the CXCR4-targeted doxorubicin encapsulated liposome (aCXCR4-Dox-LP).

Figure 2

Characterization of CXCR4 gene and surface expression on metastatic breast cancer and normal breast cancer cells. CXCR4 gene expression was quantified by qRT-PCR in Figure 2a. CXCR4 fold change is relative to GAPDH. CXCR4 surface expression was characterized in (b) HCC1500, (c) MDA-MB-175VII and (d) MCF10A cells via a flow cytometry. Blue curve represents the fluorescence of cells stained with PE-conjugated anti-CXCR4 antibody; Red curve represents the fluorescence of cells stained with PE-conjugated-IgG as controls. Figures 2d-l are representative confocal fluorescence microscope images of CXCR4 immunofluorescent staining in HCC1500 (e-g); MDA-MB-175VII (h-j); and MCF10A (k-m). DAPI was used to stained the cell nuclei; Mouse anti-human CXCR4 antibody (primary) and goat anti-mouse NL557 antibody (secondary) were used to stain CXCR4.

Figure 3

(a) Cellular binding of immunoliposomes in HCC1500, MDA-MB-175VII and MCF10A. Cells were treated with CXCR4-targeted liposome with rhodamine-dextran (aCXCR4) and IgG liposome with rhodamine-dextran (IgG, control), then characterized via flow cytometry. Figures 3(b-j) are representative confocal fluorescent microscope images of immunoliposome cellular binding in HCC1500 (b-d), MDA-MB-175VII (e-f) and MCF10A (g-j). DAPI and cell tracker green were used to stain cell nucleus and cytoplasm. CXCR4-targeted liposome with rhodamine-dextran was tested in this experiment.

Figure 4

(a) Cumulative Dox release from aCXCR4-Dox-LPs in pH 7.4 (▼) and pH 5.5 (Δ) buffers at 37°C Release of free doxorubicin in pH 7.4 (●) and pH 5.5 (○) buffers; (b and c) In vitro cell cytotoxicity of aCXCR4-Dox-LP on HCC1500 and MDA-MB-175VII. Cells were treated with free Dox (▼), CXCR4 targeted liposomes without Dox (aCXCR4-LPs) (Δ), unconjugated Dox encapsulated liposomes (unconjugated Dox-LPs) (●), or CXCR4 targeted Dox encapsulated liposomes (aCXCR4-Dox-LPs) (○) with varying Dox concentrations. Differences between aCXCR4-Dox-LPs and unconjugated Dox-LPs were analyized by student t-test. * p < 0.05; ** p < 0.01.