Induction of multiple myeloma cancer stem cell apoptosis using conjugated anti-ABCG2 antibody with epirubicin-loaded microbubbles

Abstract

Background: Multiple myeloma (MM) currently remains largely incurable. Cancer stem cells (CSCs) are believed to be responsible for drug resistance and eventual relapse. In this study, we exploited a novel agent to evaluate its inhibitory effect on MM CSCs.

Methods: Epirubicin (EPI)-loaded lipid microbubbles (MBs) conjugated with anti-ABCG2 monoclonal antibody (EPI-MBs + mAb) were developed and their effect on MM 138^-CD34^- CSCs isolated from human MM RPMI 8226 cell line plus ultrasound exposure in vitro and in vivo in a nonobese diabetic/severe combined immunodeficient mouse model were assessed.

Results: EPI-MBs + mAb combined with ultrasound led to a significant decrease in the clone formation ability and the mitochondrial membrane potential along with an increase in MM CSC apoptosis. Moreover, treatment with EPI-MBs + mAb with ultrasound exposure remarkably inhibited the growth of MM CSC-derived tumors in xenograft nonobese diabetic/severe combined immunodeficient mice compared with a single agent or EPI-MBs + mAb without ultrasound exposure. The inhibitive efficacy was also correlated with an increased expression of caspase-3, Bax, and TUNEL and decreased expressions of PCNA, Bcl-2, and CD31.

Conclusions: Our findings reveal that the EPI-MBs + mAb combined with therapeutic ultrasound may confer an effective approach for treatment of MM by induction of an apoptotic pathway in MM CSCs.

Keywords: Cancer stem cells; Epirubicin; Microbubble; Multiple myeloma; Ultrasound.

Fig. 1

Analysis of epirubicin (EPI) entering MM CSCs. The images acquired from the confocal fluorescence microscopy were analyzed with Image J software, and the fluorescence intensity of cells in EPI-MBs + mAb + US was set to 100 to provide a basis for comparison. The relative fluorescence intensity of various groups was calculated. a Representative images show EPI entering MM CD138⁻CD34⁻ CSCs (red) 30 min after cells were respectively incubated with PBS (control), EPI (10 μg/mL), and EPI-MBs + mAb + US (0.5 W/cm²) and then stained with 4’,6-diamidino-2-phenylindole (DAPI) for 10 min as described in the Methods. Blue, red, and pink fluorescence intensity represents the DAPI (cellular nucleus), EPI (entering MM CSCs), and these merged, respectively. b Quantification of red fluorescence intensity in the different treated cells. *P < 0.05, ** P < 0.01, *** P < 0.005. EPI, epirubicin; mAb, monoclonal antibody; MB, microbubble; US, ultrasound

Fig. 2

Analysis of clone formation, membrane potential, and cell cycle of MM CSCs. As described in the Methods, 1 × 10⁶ MM CSCs treated with various agents for 30 min were used for assay clone formation, membrane potential, and cell cycle analysis. a Images showing clone formation rate. c,e Changes in mitochondrial membrane potential and cell cycle were analyzed by FCM. b,d,f Statistical analysis of the clone formation rate and changes in mitochondrial membrane potential and cell cycle. *P < 0.05, **P < 0.01, ***P < 0.005. EPI, epirubicin; mAb, monoclonal antibody; MB, microbubble; US, ultrasound

Fig. 3

Detection of EPI-loaded MBs in MM tissues. a Images of tumor-bearing mice on day 34 after 1 × 10⁶ MM CD138⁻CD34⁻ cells or non-MM CD138⁻CD34⁻ cells were injected s.c. into NOD/SCID mice. b Dynamic tumor growth curve. c MBs were located in the MM tissues of mice injected with EPI-MBs + mAb (left) or EPI-MBs (right) 30 min after injection. d Quantification analysis was performed on tumor sections observed under light microscopy according to MB counts in EPI-MBs + mAb and EPI-MBs groups. e Ultraphonic echo intensity was analyzed as described in the Methods. f Ultraphonic echo intensity of statistical comparisons referring to G1 or S. *P < 0.05, *** P < 0.005. EPI, epirubicin; mAb, monoclonal antibody; MB, microbubble; ns, not significant

Fig. 4

Therapeutic effect of EPI-MBs + mAb combined with ultrasound exposure on MM CD138⁻CD34⁻ CSC-derived tumors in NOD/SCID mice (six per group). a Dynamic tumor volumes. The MM-bearing NOD/SCID mice were treated with the different agents as described in the Methods. b Quantification analysis of survival time. c Tumor histopathologic changes (hematoxylin and eosin; original magnification, ×400) in MM-bearing mice treated with the different agents. d Quantification of apoptosis. *P < 0.05, **P < 0.01, ***P < 0.005. EPI, epirubicin; mAb, monoclonal antibody; MB, microbubble; US, ultrasound

Fig. 5

Expression of apoptosis-related molecules in tumor tissues analyzed with immunohistochemistry and Western blot. a,e Images of apoptosis-related molecule expression, including Caspase-3, Bax, TUNEL, PCNA, Bcl-2, and CD31 analyzed with immunohistochemistry (a, original magnification ×400; e, original magnification ×100). b–d,f–h Quantification analysis of Caspase-3, Bax, TUNEL, PCNA, Bcl-2, and CD31 expression. i Expression of P-p65and P-IκBα analyzed by Western blot. j Semiquantification analysis of molecular expression. *P < 0.05, **P < 0.01. EPI, epirubicin; mAb, monoclonal antibody; MB, microbubble; US, ultrasound