MRI for Diagnosis and Treatment of Breast Cancer Stem Cells Using Multifunctional Nanoparticles

Abstract

Introduction: The identification of breast tumor initiating cells (BTICs) is important for diagnosis and therapy of breast cancers. This study was undertaken to evaluate whether the extra domain-B of fibronectin (EDB-FN) could be used as a new biomarker for BTICs and whether EDB-FN peptide conjugated superparamagnetic iron oxide nanoparticles (SPIONs) loaded with doxorubicin (Dox) could be used as a multifunctional magnetic resonance imaging (MRI) contrast agent for achieving simultaneous diagnostic imaging and therapeutic application of BTICs expressing EDB-FN in vitro and in vivo . Methods: The human breast cancer cell lines (MCF-7, BT-474, SUM-225, MDA-MB-231) and NDY-1 cells, which display the tumor-initiating abilities and drug resistance, isolated from the cancer tissues of a breast cancer patient were used. mRNA expressions were assessed by RT-PCR. ALDEFLUOR assay to measure ALDH1 activity was performed. A high-affinity and high-specificity peptide ligand for EDB-FN, designated as APTEDB was synthesized. APTEDB or APTscramble-conjugated TCL-SPION (APTEDB-TCL-SPION or APTscramble-TCL-SPION) were developed for selective EDB-FN-targeted MRI. NDY-1 cells were subcutaneously injected into the back right flank of female BALB/c nude mice (4-5 week old) for xenograft tumor model. In vitro and in vivo MRI studies were performed by using a 3T clinical MR scanner. For the analysis of selective EDB-FN targeting MRI of BTIC tumors, T2*-weighted images of tumor were obtained and the signal intensity changes of tumors were quantitatively analyzed before and after the intravenous injection with APTEDB-TCL-SPION or APTscramble-TCL-SPION. To examine the therapeutic efficacy of Dox-loaded TCL-SPIONs (Dox@APTEDB-TCL-SPION or Dox@APTscramble-TCL-SPION), in vitro cellular toxicity was assessed by a MTT assay and tumor volumes were measured after three times administration of Dox@APTEDB-TCL-SPION or Dox@APTscramble-TCL-SPION into tail vein. After MRI examination, the histological analysis of the several tissues and tumors isolated from mice was performed. Result: While BTICs (NDY-1) had abundant EDB-FN expressions, non-BTICs (MCF-7, BT-474, SUM-225, MDA-MB-231) did not. Transfection of the BTICs with EDB-FN siRNA significantly reduced the expression of EDB-FN and several genes related to BTIC markers, self-renewal, epithelial-mesenchymal transition and drug resistance. Furthermore, Cy3.3-labeled EDB-FN specific peptide (APTEDB) showed preferential binding to the target NDY-1 cells. To construct an EDB-FN targeting imaging probe, APTEDB was covalently attached to thermally cross-linked SPION (TCL-SPION) to yield APTEDB-TCL-SPION. In the in vitro MRI of cell phantoms, selective binding of APTEDB-TCL-SPION on NDY-1 cells was evident, but little binding was observed in MCF-7 cells. After the intravenous injection of APTEDB-TCL-SPION to the xenograft NDY-1 tumor model in mice, significant signal decrease within the tumor was observed in T2*-weighted images, but there was only a marginal signal change in the case of non-targeting SPIONs such as APTscramble-TCL-SPION or TCL-SPION. In addition cell survival and tumor growth was remarkably inhibited in the Dox@APTEDB-TCL-SPION-treated group compare with untreated group and Dox@APTscramble-TCL-SPION-treated group. Conclusions: we report, for the first time, that EDB-FN is highly expressed in BTICs but not in differentiated breast cancer cells, suggesting that EDB-FN can be used as a new characteristic biomarker for identifying BTICs. Furthermore, we developed an imaging probe for targeting BTICs, which was constructed by covalently attaching EDB-FN specific high-affinity peptide to TCL-SPION and enabled visualization of BTICs within the tumor mass in vivo by a clinical 3T MR scanner. Furthermore, Dox@APTEDB-TCL-SPION showed selective drug-delivery efficacy in the xenograft mouse model. The active tumor-targeting ability of Dox@APTEDB-TCL-SPION allows for the selective detection of EDB-FN positive BTIC tumors by MRI as well as the effective delivery of anti-cancer drugs to the target tumor sites simultaneously. In the near future, we will study the therapeutic feasibility of APTEDB-TCL-SPION combined with therapeutic drugs and EDB-FN RNAi as a novel theranostic system that allows for simultaneous diagnosis and therapy of hard-to-treat BTICs in breast cancers. The nanoparticle complexes loaded with specific ligand and therapeutic drugs and genes appears to have significant potential for the characterization of tumors and for the prediction of prognosis in the context of treatment stratification and early assessment of tumor response to therapy.