Superparamagnetic Nanoparticles-based Haracterization Of Biological Properties Of Cancer Stem Cells

Cancer stem cells (CSCs) are widely thought as the "seeds" of tumor formation, and playa crucial role in the process of tumor initiation, retention, transfer and recurrence, and areamong central points for cancer research and targeted therapy. However, the scarcity of CSCsand their difficulty for isolation and purification have become the key technical bottleneckproblem in the research of cancer stem cells. Thus to establish an effective system foridentification and isolation of CSCs is definitely of great importance in cancer research andtherapy. Therefore, in the present study, human glioblastoma multiforme U251cell line wasused to establish an efficient magnetic nanoparticles-based separation system for enrichmentand isolation of CSCs. The biological properties of CSCs potentially affected by magneticnanoparticles were further analyzed to provide a theoretical basis for potential utilization ofmagnetic nanoparticles in the isolation and purification of CSCs and cancer diagnosis andtherapy in future. According to our present results (data), the conclusions were made asfollows.1. The serum-free suspension culture in combination with the cell cycle specific drugvincristine (VCR) was applied to enrich and separate CSCs derived from human glioblastomamultiforme U251cell line. The results showed that this method could effectively enrichcancer stem cells, and the enriched CSCs exhibited a good capability of continuous culturingand self-renewal. The MTT method and the FDA/PI fluorescent double staining proved thatthe enriched CSCs induced by VCR had an excellent proliferation potential and cellularviability. The RT-PCR analysis and immunocytochemistry staining results showed that thespecific markers CD133and nestin were positively expressed in CSCs, and the glioblastomaspheres had a potential of multi-differentiation, and could differentiate into astrocytes markedwith glial fiber acid protein (GFAP), oligodendrocytes with myelin basic protein (MBP), andneurons with microtubules related proteins (MAP2and tau). The RT-PCR analysis resultsshowed multi-drug resistance-associated protein1(MRP1) and anti-apoptotic protein survivingene strongly expressed in CSCs. Therefore, the VCR induced enrichment and separation ofCSCs could be used to carry out the follow-up experimental work.2. The effects of magnetic nanoparticles on biological properties of cancer stem cells (CSCs) were analysed by revealing the performance of tumor stem cells after treatment withpoly-L-lysine (PLL) modified γ-Fe₂O₃nanoparticles including proliferation, cellular viability,self-renewal, multi-differentiation potential, cell cycle distribution and apoptosis, and so on.The partial reduction co-precipitation method was used to prepare superparamagnetic γ-Fe₂O₃nanoparticles, and the positively charged PLL was applied to modify γ-Fe₂O₃nanoparticles to improve surface absorption of target cells, and the PLL-modified γ-Fe₂O₃nanoparticles were used to surface label cancer stem cells derived from human U251cell line.The Prussian blue staining and atomic absorption spectrometry analysis showed that the PLLmodified γ-Fe₂O₃nanoparticles successfully labeled CSCs. The FDA/PI fluorescent doublestaining and MTT analysis showed that the magnetic labeling did not affect cellular viabilityand proliferation capacity of CSCs and their differentiated progeny. The immunocyto-chemistry staining and semi-quantitative RT-PCR analysis showed that the magnetic labelingdid not affect specific marker expression of cancer stem cells and their multi-differentiationpotential. Besides, the magnetically labeled glioblastoma spheres could reproduce andpropagate indicating that the magnetically labeled CSCs had an intact capability forself-renewal. In addition, flow cytometric analysis showed that the magnetic labeling andunlabeling in CSCs and differentiated progeny did not affect cell cycle distribution (cells in Sphase accounting for15.93%vs15.44%and11.35%vs11.31%), and the early apoptosisrates of magnetically labeled CSCs and their differentiated progeny were4.4%and1.4%, incontrast with the unlabeled counterparts (4.9%and1.1%). Taken together, these findingsprovided solid foundations for utilization of magnetic nanoparticles in the isolation of CSCs,cancer treatment and therapy, and noninvasive capture of CSCs by magnetic resonanceimaging in vivo etc.3. The identified peroxidase-like activity of superparamagnetic Fe₃O₄nanoparticlespotentially regulating the proliferation of cancer stem cells was analyzed by examining thelevels of reactive oxygen species (ROS). The results showed that the PLL modification ofFe₃O₄nanoparticles did not affect the peroxidase-like activity, and the PLL modified Fe₃O₄nanoparticles could lower endogenous ROS levels produced within cancer stem cells and thusto promote proliferation and accelerate cell cycles of cancer stem cells. The peroxidaseactivity of cancer stem cells treated with the PLL modified Fe₃O₄nanoparticles wassignificantly increased. In addition, the PLL modified Fe₃O₄nanoparticles did not affect thespecific marker expression of cancer stem cells, multi-differentiation potential and actinstructure within cells. Different from the previous reports, our results definitely indicated thatFe₃O₄nanoparticles could beneficially enhance cell growth, which provides powerfulevidence for possible use of Fe₃O₄nanoparticles in biological medicine field in future. 4. The high temperature-aided emulsification method was applied to prepare superpara-magnetic HSA/γ-Fe₂O₃microbeads as a carrier, and then further used specifically to captureantibody (anti-CD133monoclonal antibody) by specific affinity interaction between avidinand biotin to form as immunomagnetic beads (IMBs), which was further used to purify cancerstem cells. The results showed that the active IMBs were successfully prepared and could beeffectively used for isolation and purification of target CD133⁺cells, and the successfulseparation of CD133⁺cells could be achieved within40min. Besides, the sorted CD133⁺cellsdisplayed an intact cellular viability, and could proliferate as glioblastoma spheres. Theimmunocytostainings showed that the formed tumor spheres had a multi-differentiationpotential and could highly express their specific markers nestin and CD133.In conclusion, in the present study we have developed an effective method using aserum-free suspension culture combined with the cell cycle specific drug VCR to efficientlyenrich and isolation of cancer stem cells, and the effects of magnetic labeling bysuperparamagnetic nanoparticles on biological properties of cancer stem cells were fullyanalyzed, which definitely provided solid foundations for future utilization of magneticnanoparticles in the research of cancer stem cells. Furthermore, the peroxidase-like activity ofmagnetic Fe₃O₄nanoparticles was analyzed in regulating proliferation of cancer stem cells,and a cost-effective immunomagnetic beads-based system for isolation and purification ofcancer stem cells was successfully developed, which could be also potentially applied forisolation and purification of target cells derived from other tumor cell lines, except for humanU251cell line used here.