extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Directional Release of Reovirus from the Apical Surface of Polarized Endothelial Cells

ABSTRACT Bloodstream spread is a critical step in the pathogenesis of many viruses. However, mechanisms that promote viremia are not well understood. Reoviruses are neurotropic viruses that disseminate hematogenously to the central nervous system. Junctional adhesion molecule A (JAM-A) is a tight junction protein that serves as a receptor for reovirus. JAM-A is required for establishment of viremia in infected newborn mice and viral spread to sites of secondary replication. To determine how viruses gain access to the circulatory system, we examined reovirus infection of polarized human brain microvascular endothelial cells (HBMECs). Reovirus productively infects polarized HBMECs, but infection does not alter tight junction integrity. Apical infection of polarized HBMECs is more efficient than basolateral infection, which is attributable to viral engagement of sialic acid and JAM-A. Viral release occurs exclusively from the apical surface via a mechanism that is not associated with lysis or apoptosis of infected cells. These data suggest that infection of endothelial cells routes reovirus apically into the bloodstream for systemic dissemination in the host. Understanding how viruses invade the bloodstream may aid in the development of therapeutics that block this step in viral pathogenesis. IMPORTANCE Bloodstream spread of viruses within infected hosts is a critical but poorly understood step in viral disease. Reoviruses first enter the host through the oral or respiratory route and infect cells in the central nervous system. Spread of reoviruses to the brain occurs by blood or nerves, which makes reoviruses useful models for studies of systemic viral dissemination. In this study, we examined how reoviruses infect endothelial cells, which form the walls of blood vessels. We found that reovirus infection of endothelial cells allows the virus to enter blood vessels and serves as a means for the virus to reach high titers in the circulation. Understanding how reovirus is routed through endothelial cells may aid in the design of antiviral drugs that target this important step in systemic viral infections.

IMPORTANCE Bloodstream spread of viruses within infected hosts is a critical but poorly understood step in viral disease. Reoviruses first enter the host through the oral or respiratory route and infect cells in the central nervous system. Spread of reoviruses to the brain occurs by blood or nerves, which makes reoviruses useful models for studies of systemic viral dissemination. In this study, we examined how reoviruses infect endothelial cells, which form the walls of blood vessels. We found that reovirus infection of endothelial cells allows the virus to enter blood vessels and serves as a means for the virus to reach high titers in the circulation. Understanding how reovirus is routed through endothelial cells may aid in the design of antiviral drugs that target this important step in systemic viral infections.
tion through the blood (4). The function of sialic acid and JAM-A in reovirus infection of polarized endothelial cells is not known.
In this study, we examined reovirus infection of polarized endothelial cells to better understand mechanisms of viral entry into and egress from the bloodstream. We found that reovirus productively infects polarized endothelial cells from both apical and basolateral routes of adsorption. Infection was more efficient after adsorption from the apical surface, a property attributable to the binding of sialic acid and JAM-A. Interestingly, reovirus was released exclusively from the apical surface in a noncytolytic manner. These studies provide a new understanding of how viruses infect polarized endothelial cells and identify the endothelium as an important mediator of viral pathogenesis.

Reovirus infection of polarized endothelial cells is more efficient from the apical surface.
To determine whether reovirus productively infects polarized endothelial cells (see Fig. S1 in the supplemental material), we adsorbed either the apical or the basolateral surface of polarized human brain microvascular endothelial cells (HBMECs) with strain T3SAϩ, a virus that efficiently binds sialic acid and JAM-A (15,22). The viral titer in cell lysates increased over time, regardless of the route of adsorption (Fig. 1A). Following apical adsorption, the viral titer peaked at 24 h postinfection, with the yield reaching approximately 1,000fold over the input. In contrast, following basolateral adsorption, viral replication was delayed, with yields of 5-fold at 24 h and 100-fold at 48 h postinfection. These data indicate that reovirus infection of polarized HBMECs by either the apical or the basolateral entry route is productive, but apical adsorption results in more efficient replication and increased viral yields.
Because we observed higher peak titers in polarized HBMECs after apical adsorption, we sought to determine whether initiation of reovirus infection is more efficient when cells are infected apically than when they are infected basolaterally. Polarized HBMECs were adsorbed with virus by the apical or basolateral route, and the percentage of reovirus antigen-positive cells was quantified by flow cytometry. Apical adsorption resulted in approximately 10-fold more infected cells than did basolateral adsorption (Fig. 1B). As a control, apical or basolateral adsorption of nonpolarized L929 fibroblast cells cultivated on Transwell inserts yielded equivalent numbers of infected cells (Fig. 1B).
To determine whether differences in infectivity are attributable to differences in virus binding, we assessed virus attachment to polarized HBMECs following apical or basolateral adsorption. In concordance with the infectivity data, approximately 10-fold more virus was bound to HBMECs following apical adsorption than following basolateral adsorption (Fig. 1C). As anticipated, virus bound equivalently to L929 fibroblasts following adsorption either apically or basolaterally (Fig. 1C). Together, these data suggest that reovirus binds more efficiently to the apical surface of polarized HBMECs, which results in increased infectivity and replication.
Sialic acid and JAM-A are required for reovirus infection of polarized endothelial cells. To determine whether differences in the infectivity of polarized HBMECs after apical or basolateral adsorption are attributable to differences in receptor engagement, we used mutant reovirus strains impaired in the capacity to bind either sialic acid or JAM-A. Single amino acid mutations in the 1 attachment protein can dramatically diminish binding to these FIG 1 Reovirus infection of polarized HBMECs is more efficient following adsorption from the apical surface. Polarized HBMECs were adsorbed either apically (white bars) or basolaterally (black bars) with reovirus T3SAϩ at an MOI of 10 PFU per cell. (A) Transwell inserts were excised at 0, 24, and 48 h postinfection, and viral titers in cell lysates were determined by plaque assay. A representative experiment of three performed, with each experiment conducted in duplicate, is shown. Error bars indicate the range of data for the duplicates. (B) HBMECs were incubated for 20 to 24 h and harvested by trypsinization. Cells were permeabilized and stained with Alexa Fluorconjugated, reovirus-specific antiserum. The percentage of infected cells was determined by flow cytometry. A representative experiment of three performed, with each experiment conducted in duplicate, is shown. Error bars indicate the range of data for the duplicates. (C) HBMECs were removed immediately after adsorption and stained with Alexa Fluor-conjugated, reovirus-specific antiserum. MFI was determined by flow cytometry. A representative experiment of three performed, with each experiment conducted in duplicate, is shown. Error bars indicate the range of data for the duplicates. **, P Ͻ 0.005. receptors (15,23). Polarized HBMECs were adsorbed apically or basolaterally with wild-type or mutant reovirus strains, and the percentage of infected cells was quantified at 24 h postinfection. There were significantly more infected cells following apical adsorption with wild-type strain type 3 Dearing (rsT3D) than after apical adsorption with mutant strain rsT3D-1R202W, which is deficient in sialic acid binding (21,23), or mutant strain rsT3D-1G381A, which is deficient in JAM-A binding (24) (Fig. 2A). Treatment of polarized HBMECs with neuraminidase (to remove cell surface sialic acid) and JAM-A-specific antibody prior to apical virus adsorption significantly decreased infection by rsT3D. Similarly, neuraminidase and JAM-A-specific antibody pretreatment substantially decreased infection of polarized HBMECs by rsT3D-1G381A and rsT3D-1R202W, respectively ( Fig. 2A). Concordantly, rsT3D bound more efficiently to the apical surface of polarized HBMECs than did the mutant virus strains, and virtually all virus binding was abolished by neuraminidase or JAM-A-specific antibody pretreatment (Fig. 2C). We observed a similar trend after basolateral adsorption in that diminished receptor engagement by mutant viruses or blockade of receptor engagement with inhibitors significantly decreased the percentages of virusinfected and virus-bound cells ( Fig. 2B and D). However, the overall percentage of infected cells and levels of virus binding after basolateral adsorption were substantially lower than those following apical adsorption, which diminishes the magnitude of the observed differences (note the different y axis scales in Fig. 2C and D). Reovirus mutant rsT3D-1R202W bound to the basolateral surface of HBMECs equivalently to wild-type rsT3D but infected significantly fewer cells, suggesting that sialic acid engagement may enhance reovirus replication at a postattachment step following basolateral adsorption of polarized endothelial cells. These data suggest that infection of polarized endothelial cells is dependent on virus binding to sialylated glycans and JAM-A on the apical and basolateral surfaces of polarized endothelial cells, but binding to the apical surface is more efficient.
To determine whether increased binding of reovirus to the apical surface of polarized HBMECs is attributable to enhanced receptor expression, we examined the distribution of JAM-A on polarized HBMECs by confocal microscopy. Polarized HBMEC monolayers were stained with antibodies specific for TJ protein claudin-1, as well as JAM-A (Fig. 3A). Substantially more JAM-A staining was detected at the apical surface of the polarized cell monolayer (Fig. 3B), including nonjunction sites that lack detectable claudin-1 staining (Fig. 3A). Confocal micrographs of apical portions of cells showed a stippled pattern of JAM-A expression. In equatorial sections of cells, JAM-A was distributed at the cell periphery, presumably in contact with JAM-A on adjacent cells. In these images, TJ puncta marked by claudin-1 and JAM-A colocalization are clearly visible (Fig. 3A, white asterisks). At the basolateral surface, the JAM-A signal was diminished in intensity and diffusely localized compared with JAM-A staining at the apical surface ( Fig. 3A and B). Increased distribution of JAM-A to the apical surface of polarized HBMECs may allow reovirus to bind and infect these cells more efficiently by this route.
Reovirus is released apically from infected polarized endothelial cells. We next determined whether progeny virus is released apically or basolaterally from infected polarized endothelial cells. Polarized HBMECs were adsorbed apically or basolaterally with virus, and titers within the apical and basolateral compartments were quantified at various intervals by plaque assay. After Cells were incubated for 20 to 24 h, removed from Transwell inserts with trypsin, permeabilized, and incubated with Alexa Fluor-conjugated, reovirusspecific antiserum. The percentage of infected cells was determined by flow cytometry. A representative experiment of two performed, with each experiment conducted in duplicate, is shown. Error bars indicate the range of data for the duplicates. (C, D) Cells were harvested from Transwell inserts immediately after adsorption and stained with Alexa Fluor-conjugated, reovirus-specific antiserum. MFI was quantified by flow cytometry. Note that different y axis scales are used for apical and basolateral adsorption. A representative experiment of two performed, with each experiment conducted in duplicate, is shown. Error bars indicate the range of data for the duplicates. *, P Ͻ 0.05; **, P Ͻ 0.005. apical adsorption, the viral titer in the apical compartment increased more than 30-fold at 24 h and more than 3,000-fold at 48 h (Fig. 4A). Interestingly, no virus was detected in the basolateral compartment at any time point tested (Fig. 4A). After basolateral adsorption, virus was detected in the basolateral compartment at all of the intervals tested (Fig. 4B). However, titers did not increase over time, suggesting that infectious virus in this compartment is most likely residual virus from the inoculum. The viral titer within the apical compartment was detected at 24 h postinfection and increased approximately 100,000-fold by 48 h postinfection (Fig. 4B). Therefore, regardless of the route of adsorption, reovirus egress from polarized endothelial cells occurs from the apical surface.

Reovirus infection does not alter endothelial cell TJ integrity.
To determine whether reovirus infection alters the integrity of TJs in the polarized monolayer, we quantified the transendothelial electrical resistance (TEER) at both early and late times postadsorption. After adsorption with a multiplicity of infection (MOI) of 1,000 PFU per cell, no significant alteration in TEER was ob-  served in the 2-h postinfection interval (Fig. 5A). Similarly, after adsorption with an MOI of 10 PFU per cell, no significant alteration in TEER was observed at 1 or 2 days postinfection (Fig. 5B). We conclude from these data that reovirus does not alter the function of endothelial TJs during infection.
Reovirus egress from polarized HBMECs occurs noncytolytically. To determine whether reovirus egress from infected polarized HMBECs is associated with cell lysis, we assessed cell viability with trypan blue. Polarized HBMECs or confluent L929 cells cultured on Transwell inserts were adsorbed apically or basolaterally at an MOI of 10 PFU per cell, and cell viability was quantified at 24 h postinfection. Levels of HBMEC lysis were lower than the background levels of lysis in mock-treated HBMECs after either apical or basolateral virus adsorption (Fig. 6A). In contrast, more than half of the population of infected L929 cells was lysed at 24 h postinfection. These data suggest that reovirus infection of polarized HBMECs does not compromise cell viability. . Cells were washed, fresh medium was added to the apical and basolateral compartments, and TEER was determined at the times shown. A representative experiment of two (A) or three (B) performed, with each experiment conducted in duplicate, is shown. Error bars indicate the range of data for the duplicates. TEER from the various samples was compared by one-way ANOVA. Student's t test was used to evaluate differences between mockinfected and apically infected (A) or mock-infected and basolaterally infected (B) samples. No differences were statistically significant. Reovirus is capable of inducing apoptosis in many types of cultured cells (25)(26)(27)(28) and in the CNS of infected mice (1)(2)(3)29). Although polarized HBMECs remain intact after reovirus infection, we wondered whether reovirus egress from polarized HBMEC monolayers might occur via apoptosis. To test this hypothesis, we adsorbed polarized HBMECs apically or basolaterally at an MOI of 100 PFU per cell and quantified levels of apoptosis at 24 and 48 h postinfection by using terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. At 24 h postinfection, 17.7% of the cells were infected after apical adsorption but apoptosis was detectable in only 0.9% of those cells (Fig. 6B). At 24 h after basolateral adsorption, 3.0% of the cells were infected but apoptosis was not detected in those cells (Fig. 6B). At 48 h after apical adsorption, 29.5% of the cells were infected with reovirus, with only 3.0% showing evidence of apoptosis (Fig. 6B). After basolateral adsorption, 6.6% of the cells were infected with reovirus, yet only 1.4% of those cells were apoptotic (Fig. 6B). As a positive control, treatment of polarized HBMECs with staurosporine resulted in~50% of the cells displaying evidence of apoptosis with a concomitant decrease in TEER ( Fig. 6B and C), suggesting that the low levels of apoptosis in reovirus-infected cells are not attributable to an inherent block to apoptosis in HBMECs. These data suggest that reovirus egress from polarized HBMECs occurs without inducing apoptosis.

Many viruses cause disease in infected hosts after bloodstream spread from an initial site of infection to a distant target site.
Reoviruses are neurotropic viruses that first replicate within the small intestine and disseminate systemically via the blood, nerves, and lymphatics. Reovirus penetration of the endothelium to invade the bloodstream may occur within the intestine or lymph nodes to allow the establishment of primary viremia. To investigate reovirus infection of the endothelium, we cultured HBMECs on Transwell membranes until polarization was achieved (see Fig. S1 in the supplemental material). Although reoviruses use TJ protein JAM-A as a receptor, TEER was not altered immediately following reovirus adsorption (Fig. 5), suggesting that TJ integrity remains intact after infection. Adsorption of polarized endothelial cells either apically or basolaterally with reovirus resulted in productive infection ( Fig. 1; see Fig. S2 in the supplemental material). Interestingly, reovirus strain T3D replicated more efficiently than strain type 1 Lang (T1L) in polarized endothelial cells (compare Fig. 1; see Fig. S2). This discrepancy might be due to differences in the cell surface expression of the sialylated glycans used by the different reovirus serotypes or cell-intrinsic properties of endothelial cells that confer serotype-dependent differences in reovirus susceptibility. Regardless of the serotype, replication was more efficient when reovirus was adsorbed to the endothelial cell apical surface ( Fig. 1; see Fig. S2), and significantly more reovirus antigen-positive cells were detected following adsorption by this route (Fig. 1B; see Fig. S2). The observed increase in infectivity and replication after apical adsorption was most likely due to increased virus binding to the apical surface (Fig. 1C). The number of cells bound by virus was actually higher than the number of cells productively infected. This finding suggests that not all viral particles bound to the cell surface complete an infectious cycle, a phenomenon observed in other cell lines (30)(31)(32). Reovirus infection of polarized endothelial cells by either the apical or the basolateral route requires the engagement of sialylated glycans and JAM-A (Fig. 2). Consistent with these findings, substantially more JAM-A is distributed to the apical than to the basolateral surface of polarized HBMECs (Fig. 3). Subconfluent, nonpolarized HBMECs are substantially more susceptible to reovirus infection than are polarized HBMECs (data not shown), presumably because of higher levels of JAM-A on the cell surface and the absence of a restriction of JAM-A expression to TJs.
Regardless of the route of adsorption, reovirus egress from infected polarized HBMECs occurs solely from the apical surface ( Fig. 4; see Fig. S2 in the supplemental material). Similarly, reovirus infection of polarized human airway epithelial cells results in apical release of progeny virions (33). Although TEER did not change appreciably over a time course of reovirus infection of HBMECs (Fig. 5), we questioned whether infected cells are extruded from the monolayer in a manner analogous to epithelial cell turnover (34). If they are, we would expect TEER to be maintained despite the detection of an increased number of nonviable cells over time. To test this hypothesis, we used trypan blue staining to determine whether polarized HBMECs infected with reovirus are lysed. Compared with infected L929 cells, which display substantial cytopathic effect after reovirus infection (28) (Fig. 6A), polarized HBMECs infected with reovirus apically or basolaterally do not undergo cell lysis (Fig. 6A), despite the presence of high viral titers in cells and supernatants ( Fig. 1 and 4). Sonication of supernatants harvested from the apical surface of polarized HBMECs did not lead to an increased viral titer, suggesting that released virus was not trapped within extruded cells or membrane-bound vesicles (data not shown). Apical or basolateral adsorption of polarized HBMECs with reovirus led to an increase in reovirus antigen-positive cells, but the number of apoptotic cells did not increase above that in mock-treated samples (Fig. 6B). Additionally, levels of apoptosis in reovirus-infected HBMECs were lower than in mock-infected cells by the complementary acridine orange and annexin V staining assays (see Fig. S3 in the supplemental material). We conclude from these data that regardless of the route of entry, reovirus release occurs from the apical surface in a manner that maintains cell viability. Because infection of polarized endothelial cells is noncytolytic, clearance of reovirus from an infected host may require cytotoxic T lymphocyte-mediated immunity in addition to neutralizing antibodies (35)(36)(37)(38)(39).
Virus infection of endothelial cells may serve as an additional mechanism to produce and maintain high levels of viremia. For example, dengue virus infection of endothelial cells leads to hightiter viremia by inducing endothelial cell apoptosis, resulting in endothelial barrier dysfunction and vascular leakage (40). Murine cytomegalovirus primarily infects hepatocytes, but virus produced from infected hepatic endothelial cells is responsible for dissemination to other organs (41,42). Similarly, reovirus may use the endothelium as a means to amplify to high titers in the bloodstream (Fig. 7). Reovirus infection from the basolateral route is not efficient, but progeny viral particles are efficiently transported to and released from the apical surface of polarized endothelial cells. Once released, progeny virions have access to the apical surface of adjacent endothelial cells and can enter those cells efficiently. This cycle may serve as a mechanism to generate high titers of virus in the bloodstream, which are observed during reovirus infection (4,10,21). Sialylated glycans and JAM-A are required for the infection of endothelial cells by both the apical and basolateral routes, which may account for the markedly diminished viremia in reovirus-infected JAM-A-deficient mice (4).
How reovirus exits the bloodstream is not clear from our study. Because JAM-A is present on the surface of hematopoietic cells, it is possible that reovirus-infected hematopoietic cells transport the virus from the bloodstream to sites of secondary replication, including the CNS. It also is possible that cells adjacent to blood vessels become infected as a consequence of infection of the endothelium. Epstein-Barr virus (EBV) binding to B cells leads to conjugate formation between B cells and epithelial cells, resulting in EBV entry into epithelial cells (43,44). Blood vessels in the brain closely appose pericytes and astrocytes, and reovirus infection of endothelial cells may induce modifications of these cells, resulting in invasion of the CNS.
Bloodstream spread is an important step in the pathogenesis of many viral diseases, but the mechanisms used by viruses to gain entry into the bloodstream are not well understood. Our work describes how viral infection of endothelial cells may allow access to and amplification within the circulation. We show that reovirus productively infects polarized endothelial cells by both the apical and basolateral routes. Infection after apical adsorption is more efficient than basolateral adsorption because of increased utilization of sialic acid and JAM-A at the apical surface. Reovirus release from polarized endothelial cells occurs exclusively from the apical surface in a manner that maintains TJ integrity and cell viability. Since TJ proteins are used as receptors by a diverse array of viruses, including adenovirus (45), feline calicivirus (46), hepatitis C virus (47,48), and several picornaviruses (45,49), our findings may provide a more general understanding of how viruses establish viremia for bloodstream spread. Moreover, the apical release mechanism employed by reovirus may be similarly generalizable, providing a potential new target for a host-specific, broadspectrum antiviral therapeutic.
The immunoglobulin G (IgG) fraction of a rabbit antiserum raised against strains T1L and T3D (31) was purified by protein A-Sepharose as previously described (9,15). Reovirus-specific IgG was conjugated to Alexa Fluor 647 with an APEX antibody labeling kit (Invitrogen). JAM-A-specific monoclonal antibody J10.4 (provided by Charles Parkos, Emory University) and claudin-1-specific antibody ab15098 (Abcam) were used in confocal microscopy imaging experiments. Alexa Fluorconjugated antibodies (Invitrogen) were used as secondary antibodies.
TEER measurements. TEER across polarized HBMEC monolayers was quantified at 3 and 6 days postseeding, on the day of infection, and at various intervals postinfection with an EVOM voltohmmeter and an EndOhm-6 cup electrode (World Precision Instruments). TEER readings for test samples were normalized by subtracting the TEER of blank collagen-coated Transwell inserts. The data are presented as unit area resistance (⍀·cm 2 ) (54).
Virus assays. Polarized HBMECs cultivated on Transwell inserts were adsorbed with virus apically or basolaterally at an MOI of 10 PFU per cell. For apical adsorption, 30 l of virus inoculum was added to the apical compartment. For basolateral adsorption, the Transwell insert was inverted in a sterile dish and 30 l of virus inoculum was added to the basolateral surface. In some experiments, cells were treated with medium, anti-JAM-A antibody (20 g/ml), or Arthrobacter ureafaciens neuraminidase (80 mU/ml; MP Biomedicals) prior to virus adsorption. After adsorption of virus at room temperature for 1 h, cells were washed twice with phosphate-buffered saline (PBS) and 200 l of medium was added to the apical compartment and 1 ml of medium was added to the basolateral compartment. For viral release assays, medium from the apical or baso- lateral compartment was collected at various intervals and viral titers in medium from each compartment were determined by plaque assay with L929 cells (37). For viral replication assays, Transwell membrane inserts were removed from Transwell inserts with a scalpel, submerged in 500 l of medium, and subjected to two cycles of freezing and thawing. Viral titers in cell lysates were determined by plaque assay with L929 cells (37). For infectivity studies, cells were incubated at 37°C for 20 to 24 h, harvested with 0.05% trypsin-EDTA (Invitrogen) at room temperature, and quenched with medium collected from the apical compartment of the respective sample. Cells were stained with Alexa Fluor-conjugated, reovirus-specific antiserum as previously described (50). The percentage of reovirus antigen-positive cells was determined by flow cytometry. For binding studies, cells were detached from the Transwell insert immediately after adsorption with Cellstripper (Mediatech) at 37°C for 5 min and stained with Alexa Fluor-conjugated, reovirus-specific antiserum as previously described (50). The mean fluorescence intensity (MFI) of each sample was determined by flow cytometry. All cell staining was quantified with FlowJo software (Tree Star).
Cell imaging. Polarized HBMECs were fixed in 100% methanol at Ϫ20°C for 5 min. Cells were blocked in PBS containing 5% bovine serum albumin at room temperature for 30 min. Cells were stained with antibodies specific for JAM-A (1:1,000) and claudin-1 (1:100) as previously described (50,55). After staining, Transwell membranes containing cells were excised with a scalpel. Membranes were placed onto glass slides, and glass coverslips (#1.5; Thermo Scientific) were mounted with Aqua-Poly/ Mount mounting medium (Polysciences, Inc.). Cell images were captured with a Zeiss LSM 510 Meta laser-scanning confocal microscope with a 63ϫ/1.40 Plan-Apochromat objective lens. A standard threshold pixel intensity was used for all images, and the pinhole size used was the same for all fluorophores. Images represent a single section or a series of sections from within a z stack and were adjusted for brightness and contrast to the same extent. The MFI of pixels from apical and basolateral sections of cells (n ϭ 5) was quantified with ImageJ software (NIH).
Trypan blue exclusion assay. HBMECs and L929 cells were cultured on Transwell inserts until polarized and confluent, respectively. Virus was adsorbed apically or basolaterally at an MOI of 10 PFU per cell, and cells were incubated at 37°C for 20 to 24 h. After incubation, cells were harvested with trypsin-EDTA, quenched with medium collected from the apical compartment, and washed once with PBS. A small aliquot (20 l) of cells was removed for analysis of cell lysis. An equal volume of trypan blue (0.4% [wt/vol] in PBS; Mediatech) was added to cells, which were then incubated at room temperature for 3 min. Lysed and intact cells were enumerated using a hemocytometer with bright-field microscopy. The percentage of reovirus-infected cells in the remainder of each sample was quantified by flow cytometry.
TUNEL assay. Polarized HBMECs were adsorbed with virus at an MOI of 100 PFU per cell, washed twice with PBS, and incubated at 37°C for 24 or 48 h. Cells were removed from the Transwell insert with trypsin-EDTA, quenched with medium collected from the apical compartment, washed once with PBS, and assayed for the percentage of apoptotic cells by the TUNEL technique (APO-BrdU TUNEL assay kit; Invitrogen) according to the manufacturer's instructions. After TUNEL staining, cells were stained with Alexa Fluor-conjugated, reovirus-specific antiserum (1:1,000) at 4°C for 30 min, washed, and pelleted. The samples were resuspended in 0.5 ml propidium iodide-containing buffer. Stained cells were analyzed for apoptosis and the presence of reovirus antigen by flow cytometry. See Text S1 in the supplemental material for the additional methods used.
Statistical analysis. Experiments were performed in duplicate and repeated at least twice. Representative results of single experiments are shown. Mean values were compared with an unpaired Student's t test or one-way analysis of variance (ANOVA) (GraphPad Prism). Error bars denote the range of data or standard deviation. P values of Ͻ0.05 were considered statistically significant.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Quantum dots incorporated magnetic nanoparticles for imaging colon carcinoma cells

Background Engineered multifunctional nanoparticles (NPs) have made a tremendous impact on the biomedical sciences, with advances in imaging, sensing and bioseparation. In particular, the combination of optical and magnetic responses through a single particle system allows us to serve as novel multimodal molecular imaging contrast agents in clinical settings. Despite of essential medical imaging modalities and of significant clinical application, only few nanocomposites have been developed with dual imaging contrast. A new method for preparing quantum dots (QDs) incorporated magnetic nanoparticles (MNPs) based on layer-by-layer (LbL) self-assembly techniques have developed and used for cancer cells imaging. Methods Here, citrate - capped negatively charged Fe3O4 NPs were prepared and coated with positively - charged hexadecyltrimethyl ammonium bromide (CTAB). Then, thiol - capped negatively charged CdTe QDs were electrostatically bound with CTAB. Morphological, optical and magnetic properties of the fluorescent magnetic nanoparticles (FMNPs) were characterized. Prepared FMNPs were additionally conjugated with hCC49 antibodies fragment antigen binding (Fab) having binding affinity to sialylated sugar chain of TAG-72 region of LS174T cancer cells, which was prepared silkworm expression system, and then were used for imaging colon carcinoma cells. Results The prepared nanocomposites were magnetically responsive and fluorescent, simultaneously that are useful for efficient cellular imaging, optical sensing and magnetic separation. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) revealed that the particle size is around 50 nm in diameter with inner magnetic core and outer CdTe QDs core-shell structure. Cytotoxicity test of prepared FMNPs indicates high viability in Vero cells. NPs conjugated with anti cancer antibodies were successfully labeled on colon carcinoma cells (LS174) in vitro and showed significant specificity to target cells. Conclusion The present report demonstrates a simple synthesis of CdTe QDs-Fe3O4 NPs. The surface of the prepared FMNPs was enabled simple conjugation to monoclonal antibodies by electrostatic interaction. This property further extended their in vitro applications as cellular imaging contrast agents. Such labeling of cells with new fluorescent-magneto nanoprobes for living detection is of interest to various biomedical applications and has demonstrated the potential for future medical use.

Background
The merging of dual natured components of optical and magnetic properties on nanoscale regime can enable new advance in molecular imaging and medical theranosis that are essential for early detection and rapid treatment of diseases [1,2]. Magnetic nanoparticles (MNPs), e.g. Fe 3 O 4 NPs, are of immense importance in the emerging area of nanomedicine because of their ability to be manipulated and detected with an external magnetic field. They can be used in numerous applications, both in vitro and in vivo, such as magnetic resonance imaging (MRI) contrast enhancement [3], bioseparation [4], biosensing [5], cancer therapy using magnetic hyperthermia [6], and targeted drug delivery [7]. Although MNPs appear to be the currently preferred cell-labeling materials, the poor signal intensity on MRI limits their clinical utility [8]. MNPs label is a major limitation for long-term tracking, as the magnetic resonance signal is lost over time due to cellular proliferation, especially with rapidly dividing cells. Hence, more efficient cellular-internalizing methods are highly preferable. Regardless of magnetic imaging, optical techniques offer improved high spatial resolution allowing the visualization of cell structures. They are used for the simultaneous visualization of multiple modalities with two or more fluorescent probes with different spectra, but have limited depth of imaging and poor absolute quantitative accuracy due to the absorption of light in tissues. Magnetic imaging has no practical limitation in terms of the depth of imaging; however, spatial resolution is poor and imaging with more than one probe is problematic [9]. Therefore, it is conceivable that one single agent to provide imaging in multiple imaging modalities (optical and magnetic) would be of great value to offer more comprehensive diagnostic information and the dynamics of disease progression. An example of such multifunctional NPs are FMNPs that bear two attractive features, fluorescence and superparamagnetism, allowing their intracellular movements to be controlled using magnetic force and monitored using a fluorescent microscopy, simultaneous delivery of fluorescence and magnetic particles to individual cells offers the opportunity of correlating optical images and MRI [8,10]. These features could lead to effective multifunctional drug-loaded MNPs that would facilitate increased drug transport rates, mucus penetration, antibiotic efficiency in biofilms and cellular imaging.
Semiconductor nanocrystals called quantum dots (QDs) is well known promising candidate as fluorescent materials for its high photostability, high emission quantum yield, narrow emission peak, size dependent wavelength tunability in comparison with organic dyes and fluorescent proteins. These properties of QDs make them more interesting for potential biomedical application such as protein trafficking, DNA detection, cellular imaging and dynamic studies of cell motility. QDs owing to their inorganic nature, are more robust than organic dyes and therefore their reduced photobleaching under light allows for real-time monitoring of biological events over extended periods of time [11,12]. Taking the advantages of QDs and MNPs, we designed, prepared and characterized new dual-marker particles, simultaneously combining both fluorescence nanocrystals, such as QDs and superparamagnetic nanoparticles (Fe 3 O 4 NPs) in one entity. Considerable research has been devoted to the combination of magnetic and fluorescent properties in a single nanocomposites [1,2,13]. However, many of those synthetic processes usually involve complexed multi-step reactions [14][15][16]. Alternatively, a rapid onepot self-assembly synthesis using controlled electrostatic force may improve photostability and colloidal stability. The cytotoxicity of prepared NPs was investigated in Vero cells using the trypan blue in vitro assay. The result indicates the cytotoxicity of FMNPs were reduced with dilution and allowed its potential use in biomedical application. The prepared FMNPs were further applied to cancer cell imaging using monoclonal antibodies against specific markers on cancer as a tool for therapy. Humanized monoclonal antibody CC49 (hCC49) [17] is a clinically validated antibody to target tumor-associated glycoprotein-72 (TAG-72) a well-known marker in colon carcinoma [18,19].
In this work, fragment antigen binding (Fab) region of hCC49 was conjugated with FMNPs, which was used for specific cancer cell imaging. Fluorescence microscopy showed significant preferential binding of the NPs conjugates by cells. Such a nanoprobe could potentially be used to image resections of cancer cells in real time and to correlate preoperative diagnostic images with intraoperative pathology at cellular-level resolution.

Spectroscopic study of CdTe QDs and FMNPs
The absorbance and photoluminescence (PL) intensity of QDs are shown in Figure 1A. The absorbance shoulder of QDs was located at 502 nm, while the PL peak of QDs was situated at 522 nm. According to Peng's equation [20], the particle size of QDs was about 2.85 nm and concentration was 2.17 μM.
Fluorescence emission spectrum of FMNPs was recorded for the supernatant and residue after magnetic separation from the reaction mixtures. Residual solution remained strong fluorescence intensity while supernatant had ignorable PL intensity ( Figure 1B). This indicates most of QDs capped on MNPs. Such a stable PL property is favorable for labeling cancer cells [8]. It is worth noting that the PL peak position (at 540 nm) for FMNPs in solutions redshifted [8] compared with free QDs, suggesting that some free NPs may aggregate to form clusters through partial electrostatic force. The clustering of individual QDs causes slight degradation of energy level, leading to narrower energy band gap and band broadening. However, it did not observe any further uncontrolled aggregation of QDs that may bring fluorescence quenching in the experiments because of strong repulsion forces among QDs through their highly negative surface charges.  Figure 2C). Taking together these results smaller size CdTe QDs covered the surface of core Fe 3 O 4 NPs. CTAB (not shown) between QDs and Fe 3 O 4 NPs will avoid NPs to come in contact of each other, which would be helpful for better fluorescence emission. Due to the strong electrostatic interaction between the negatively charged -COOgroups on the surface of the CdTe QDs and the positively charged quaternary amino groups of the CTAB, some QDs are forced to aggregate and forming a thick shell. It is better to notify that the size of NPs in TIRF image is bigger than TEM image. Usually, TIRF microscope has limitation to measure small particle size. In this  study, TIRF microscope specially used to observe core (dark) and shell (green) structure of FMNPs.

Superconducting quantum interference device (SQUID) study of FMNPs
The FMNPs were prepared by encapsulating inorganic magnetic particles covered by inorganic QDs. The MNPs were coated with CTAB and CdTe QDs using selfassembly method. Both the Fe 3 O 4 MNPs and FMNPs exhibited typical superparamagnetic behavior due to no hysteresis. The remanence and coercivity are zero, illustrating that the particles respond magnetically to an external magnetic field, and redisperse rapidly when the magnetic field is removed. Figure 3 shows hysteresis loops of the proposed MNPs and FMNPs, which indicate that they possess a magnetic saturation value of about 71.0 and 65.0 emug -1 . This large saturation magnetization makes them very susceptible to magnetic fields. The decrease in the overall magnetization values indicates that the Fe 3 O 4 surface is covered with nonmagnetic materials such as mostly QDs and some organic materials of CTAB and stabilizers. These FMNPs have superparamagnetism, and no magnetism remains without the magnetic field. These properties make the FMNPs a useful tool for micro-separation in fluidic systems.

Stability of FMNPs and cell viability in the presence of FMNPs
As far as the stability of FMNPs, the FMNPs are in aqueous solution at room temperature for > 1 month. PL intensity of FMNPs did not deteriorate until 4 weeks ( Figure 4A). This superior colloidal stability of FMNPs results from the cationic surfactant layer between QDs and MNPs which avoids QDs to come in close proximity to MNPs and retained its optical properties. FMNPs can be precipitate using external magnetic field and redispersed in water. This type of precipitationredispersion can be repeated many times, suggesting that NPs structures are very robust in nature. Fluorescence images of FMNPs during washing and cell labeling are also shown stable optical properties ( Figure 4B).
FMNPs are toxic for Vero cells, but dilution of FMNPs led to lower cytotoxicity. About 64 times diluted solution of FMNPs has shown no cytotoxicity ( Figure 4C). Compared with TGA-capped CdTe QDs, the as-prepared QDs showed lower cytotoxicity under the same conditions (data not shown). Generally, CdTe QDs are highly toxic for cells due to the release of Cd 2+ ions [21]. However, the prepared FMNPs reduce the cytotoxicity of QDs to a small extent. A reasonable interpretation is that the outer surface of FMNPs was covered by quaternary ammonium groups and leading to less released Cd 2+ ions.

Confirmation of FMNPs conjugated with Fab region of hCC49 antibody
FMNPs conjugated with Fab region of hCC49 antibodies are confirmed with bead enzyme-linked immunosorbent assay (ELISA). Higher signal was observed with Fab antibody modified FMNPs than only FMNPs ( Figure 5), suggesting that the FMNPs was successfully conjugated with antibodies.

Cellular imaging of colon carcinoma cells using FMNPs conjugated with Fab region of hCC49 antibody
Specific binding between FMNPs and cancer cells was observed by confocal laser scanning microscopy in vitro. When LS174T cancer cells were incubated with FMNPs conjugated with Fab region of hCC49 antibodies, green fluorescence around the nucleus ( Figure 6D) confirming Fab region of antibodies bound with sialylated sugar chain in TAG-72 region of LS174T cancer cells. As a negative control, LS174T cancer cells were incubated without hCC49 antibodies Fab region-conjugated FMNPs. However, negative cells show only nucleus staining by 4′,6diamidino-2-phenyindole (DAPI) (Figure 6E), no green fluorescence around the nucleus. For more specificity, HEK293 cells were incubated with FMNPs conjugated with Fab region of hCC49 antibodies and merged image of DAPI and fluorescence confirmed no green fluorescence around the nucleus ( Figure 6C and F). This indicates that HEK293 cells have no specific binding site of Fab region of hCC49 antibodies. Cell morphology was maintained after being incubated with the FMNPs, which implies that the nanocomposites had no cytotoxic effect on the cancer cells. In addition, these confocal images confirmed that the NPs were bound the cell surface and the NPs accumulated uniformly around cell surface.

Conclusion
In conclusion, dual modal fluorescent-magnetic nanocomposites based on fluorescent CdTe QDs and Fe 3 O 4 MNPs were developed by a simple LbL fabrication. This self-assembly technique retains the magnetic and fluorescence properties of FMNPs. FMNPs had good optical properties, and decent saturation magnetization. Further in vitro studies showing less toxicity at 64 fold dilutions and the ability to interact with cancer cells showing the ability of the FMNPs probes for imaging. Ultimately, it is believed that these particles will provide a new class of multimodal NPs for the complex biologic systems.

Synthesis of CdTe QDs
CdTe-QDs has been synthesized as previously reported [22]. Shortly, 0.985 g (2.35 mmol) of Cd(ClO 4 ) 2 · 6H 2 O was dissolved in 125 ml of water, and 5.7 mmol of the TGA added under stirring, followed by adjusting pH to appropriate values (11.4-11.6) by dropwise addition of 1 M NaOH. The solution was placed in a three-necked flask and deaerated by nitrogen gas-bubbling for 30 min. Under stirring, H 2 Te gas (generated by the reaction of 0.

Synthesis of Fe 3 O 4 NPs
The preparation of citrate-capped Fe 3 O 4 NPs was synthesized using the previously described method with minor modification [23]. The reaction mixture contained 1.622 g of FeCl 3 · 6H 2 O and 0.994 g of FeCl 2 · 4H 2 O, were mixed with 40 ml of distilled water. Five ml of concentrated ammonia aqueous (28%) was added in this solution and heated at 90°C. Then, 4.4 g sodium citrate was added and continued the reaction for 30 min. The MNPs thus obtained were washed with anhydrous ethanol and redispersed in deionized water for further use. Particle size and electric charge the prepared Fe 3 O 4 NPs were measured by dynamic light scattering (ZetaSizer Nano-ZS, Malvern, UK).

Preparation of FMNPs
New synthesis method for fluorescent-magnetic nanocomposites structure were prepared based on simple and efficient LbL self-assembly method [24]. Briefly, 100 ml Fe 3 O 4 NPs were dispersed in 5 ml of CTAB (10 mg/ml) solution under vagarious stirring. Next, 200 ml of ammonia solution was added and the mixture was stirred at room temperature for 30 min. Cationic surfactant CTAB capped Fe 3 O 4 NPs by electrostatic force at this stage. Then, 200 ml of CdTe QDs was added and mixtures were stirred at room temperature for 2 h for binding of QDs with CTAB. The resulting FMNPs were collected by external magnetic field, washed three times with MilliQ water and redispersed in MilliQ water for further use. The PL spectra of FMNPs were recorded using a Tecan infinite M 200 spectrophotometer. The samples were excited at 380 nm, and the exciting slit and the emission slit were 5 and 10 nm, respectively.

Trypan blue test
The cytotoxicity of the FMNPs were determined using the trypan blue exclusion test, which determines the number of viable cells present in a cell suspension. In 96-well plates, 100 μl suspension of Vero cells (5 × 10 4 cells/ml) in 5%(v/v) horse serum containing minimum essential medium was added per well and incubated in a 5% CO 2 humidified incubator at 37°C for 24 h. Then different diluted solutions of FMNPs were added to each well and further incubate at 37°C for 24 h. After medium was removed, 0.25% trypsin was added in all wells. The plate was centrifuged (Centrifuge 5430, Eppendorf AG, Germany), removed supernatant and redispersed in 5% (v/v) horse serum minimal essential medium. The 96well plates were further incubated for 15 min. Fifteen microliter of trypan blue was added to each cell well. The resulting mixture was gently shaken for 10 min at room temperature. Cell numbers were counted using haemocytometer (Line Seiki, Tokyo, Japan) under microscope (Model CHT, Olympus optical Co. Ltd., Tokyo, Japan) and the percentage of viability was calculated.

Preparation of hCC49 antibody Fab regions
The gene of hCC49 antibody [19] was amplified from plasmid pDong1 (hCC49), which is a gift from Professor Hiroshi Ueda of Chemical Resources Laboratory of Tokyo Institute of Technology by PCR using primers bx-FLAG-hCC49VHCH-F (5'-CACCATGAAGATACT CCTTGCTATTGCATTAATGTTGTCAACAGTAATG TGGGTGTCAACATGGAGCCACCCGCAGTTCGAAA AGATGAAATACCTATTG-3') and hCC49VHCH-R (5'-C TATGCGGCCCCATTCAG-3') for hCC49VHCH1, primers bx-FLAG-hCC49VLCL-F (5'-CACCATGAAGATACTCCT TGCTATTGCATTAATGTTGTCAACAGTAATGTGGG TGTCAACAGACTACAAGGATGACGATGACAAGGA TATTGTGATGACC-3') and hCC49VLCL-R (5'-TCAC TCTCCCCTGTTGAA-3') for hCC49VLCL. The amplified genes were inserted into pENTR/D-TOPO (Invitrogen), respectively, to make pENTR/D-hCC49VHCH1 and pENTR/ D-hCC49VLCL by the topoisomerase reaction. Using the above plasmids, the gene was inserted into pDEST8 using Gateway technology (Invitrogen) to construct pDEST-hCC49VHCH1 and pDEST-hCC49VLCL, which were then used to transform E. coli BmDH10Bac to obtain the recombinant Bombyx mori nucleopolyhedrin (BmNPV) bacmids, rBmNPV-hCC49VHCH1 and rBmNPV-hCC49VLCL. The recombinant bacmids (10 μg each) were mixed with onetenth volume of DMRIE-C (Invitrogen) and incubated at room temperature for over 45 min. Fifty microliters of this mixture was injected into a silkworm larva on the first day of the fifth instar larvae (Ehime Sansyu Co. Ltd., Ehime, Japan). Injected silkworm larvae were reared for 5-7 days, and the hemolymph collected was centrifuged to remove hemocytes at 2400 × g for 10 min at 4°C. The supernatant was used as a hemolymph sample for purification. Fab antibodies were purified with an ANTI-FLAG M2 Affinity Gel (Sigma) according to the instructions provided by the manufacturer.

Conjugation of hCC49 antibody Fab region with FMNPs and its binding activity
To apply FMNPs on bioimaging, the NPs were conjugated with hCC49 antibodies Fab region to assess their potential as fluorescent probes for LS174T cancer cells. Fab region of hCC49 antibodies has binding affinity with sialylated sugar chain in TAG-72 region of LS174T cancer cells. Electrostatic interaction between positively charged FMNPs (+18.3 mV, measured by ZetaSizer, Nano-ZS, Malvern, UK) and negatively charged of hCC49 antibodies (−3.53 mV, Tris glycin at pH 8.5, measured by ZetaSizer, Nano-ZS, Malvern, UK) was used to bind each other. NPs were dissolved in hCC49 antibodies (final concentration of 20 μg/ml) and kept at 10°C for 2 h with continuous stirring. To check whether antibodies were bound with FMNPs or not, at first FMNPs were blocked with 2% bovine serum albumin (BSA). After incubation at room temperature for 2 h, samples were washed three times with PBS buffer solution and redispersed in anti-FLAG antibodies conjugated horseradish peroxidase (Sigma, USA) with a final concentration of 1 ng/ml. After incubation at room temperature for 1 h, samples were washed three times with PBS buffer solution and transferred to a new tube. Signal was developed with 100 μl 3,3' ,5.5'tetramethylbenzidine (TMBZ, Sigma) substrate solution (20 μg ml -1 TMBZ and 0.2 μl H 2 O 2 in 1 ml NaOAc, pH 6.0) for 5-30 min at 25°C. The reaction was stopped by adding 100 μl of 10% H 2 SO 4 and absorbance of reaction supernatant was recorded at 450 nm with a reference at 655 nm using a micro plate reader (Model 680, Bio-Rad, Hercules, CA, USA).

Cellular imaging using hCC49 antibody Fab region conjugated FMNPs
Colon carcinoma cancer cells (LS174T) were seeded on slide glass 24 h prior to labeling and staining. LS174T cells were cultured with hCC49-conjugated NPs, without hCC49-conjugated NPs and HEK293 cells were cultured with hCC49-conjugated NPs for 24 h in a 37°C humidified incubator maintained at 5% CO 2 . After incubation, fixation of the cells were carried out using 10%(v/v) formalin for 20 min and rinsed four times using PBS (pH 7.5). Then, 50 mmol NH 4 Cl solutions were added and washed four times with PBS (pH 7.5). Next, 4% BSA solution was used as a blocking agent for 1 h at RT. Following labeling, the slide glasses were washed four times PBS buffer (pH 7.5). After washes, cellular nuclei were stained with 1% (v/v) solution of DAPI in 2% BSA solution in PBS buffer (pH 7.5) for 1 h and washed with PBS (pH 7.5) four times. Confocal images were acquired using a confocal laser scanning microscope (LSM 700, Carl Zeiss Microimaging GmbH, 07740 Jena, Germany) and Image processing was performed using LSM Software ZEN 2010.


extfieldsofstudy: Medicine Biology

text:
Understanding and targeting cancer stem cells: therapeutic implications and challenges

Cancer stem cells (CSCs) have been identified as rare cell populations in many cancers, including leukemia and solid tumors. Accumulating evidence has suggested that CSCs are capable of self-renewal and differentiation into various types of cancer cells. Aberrant regulation of gene expression and some signaling pathways has been observed in CSCs compared to other tumor cells. CSCs are thought to be responsible for cancer initiation, progression, metastasis, recurrence and drug resistance. The CSC hypothesis has recently attracted much attention due to the potential for discovery and development of CSC-related therapies and the identification of key molecules involved in controlling the unique properties of CSC populations. Over the past several years, a tremendous amount of effort has been invested in the development of new drugs, such as nanomedicines, that can take advantage of the “Achilles' heel” of CSCs by targeting cell-surface molecular markers or various signaling pathways. Novel compounds and therapeutic strategies that selectively target CSCs have been identified, some of which have been evaluated in preclinical and clinical studies. In this article, we review new findings related to the investigation of the CSC hypothesis, and discuss the crucial pathways involved in regulating the development of CSC populations and the advances in studies of drug resistance. In addition, we review new CSC-targeted therapeutic strategies aiming to eradicate malignancies.

Introduction
What are cancer stem cells (CSCs)? Recent studies have suggested that CSCs are immortal tumor-initiating cells that can self-renew and have pluripotent capacity [1] . CSCs have been identified in multiple malignancies, including leukemia and various solid cancers. Due to their extraordinary characteristics, CSCs are thought to be the basis for tumor initiation, development, metastasis and recurrence. In 1963, Bruce et al observed that only 1%-4% of lymphoma cells (not all cancer cells) can form colonies in vitro or initiate carcinoma in mouse spleen [2] . However, the first compelling evidence proving the existence of CSCs is generally acknowledged to have been provided by Bonnet and Dick in 1997 [3] . In their reports, only the CD34 + CD38cells from acute myeloid leukemia (AML) patients could initiate hematopoietic malignancy in NOD/ SCID mice. Importantly, this cell population possessed the ability to self-renew, proliferate and differentiate [3] . The first report of CSCs in solid cancer came in 2003 from Al-Hajj, who demonstrated the presence of CSCs in breast cancer [4] . To date, CSCs have been discovered in a broad spectrum of solid tumors, including lung cancer [5] , colon cancer [6] , prostate cancer [7] , ovarian cancer [8] , brain cancer [9] , and melanoma [10] , among others.

The controversial cancer stem cell models
Most tumors are thought to arise from one single cell that develops into a heterogeneous population. However, two distinct models have been proposed to account for tumor growth and the heterogeneity within tumors. In the cancer stem cell model, the modalities of cancer, such as initiation, progression, metastasis and recurrence, depend primarily on rare stem cells. The heterogeneity and hierarchy between all of the cells within a tumor result from asymmetric division of CSCs. This model suggests that tumors are highly hierarchical with a unique self-renewing population of cells at the top of the hierarchy. All other cells comprising the tumor bulk are derived from differentiated CSCs [11] .
However, an alternative view also exists, called the clonal evolution model (CE model) [12] . This model posits that all tumor cells contribute to tumor maintenance with differing capacities. The intercellular variation is primarily attributed to subclonal differences that result from genetic and/or epigenetic changes during cancer development. Generally speaking, the CSC model emphasizes the concept of functional heterogeneity without considering the existence of intracellular genetic variation or genetically diverse subclones. In contrast, the CE model focuses on genetic heterogeneity but ignores the functional variation within individual genetic subclones.
Although these two models seem to be mutually exclusive [13] , accumulating evidence has suggested that neither the CSC model nor the CE model should be rejected. Recently, several groups have combined functional assays with genetic analysis to examine the genetic diversity of tumor propagating cells or tumor initiating cells in both leukemia and solid cancer [14][15][16] . Anderson and co-workers first directly proved the existence of genetic diversity of cancer propagating cells within individual ETV6-RUNX1-positive acute lymphoblastic leukemia (ALL) patients [14] . In addition, the genetic diversity and relative dominance of subclones vary with the development of disease [14] . Similarly, Notta et al have established that individual Bcr-Abl + ALL samples are composed of genetically distinct subclones that are related by a complex evolutionary process [15] . These authors also determined that genetically diverse subclones already possess variably aggressive properties at the time of diagnosis [15] .
These studies have indicated that cancer stem cells exist but might evolve over time. An ancestral clone gives rise to at least two clonal lineages that evolve independently, with each clone acquiring diverse genetic aberrations; one clone emerges as the dominant diagnostic clone, while the other clone gives rise to the predominant clone containing additional mutations at relapse [15] .

Characteristics of cancer stem cells
CSCs are distinct populations of tumor cells. CSCs have multiple unique features that cause them to be vital for tumor formation. CSCs can self-renew [17] and are immortal and necessary to maintain the population of tumor cells. CSCs are pluripotent and can generate tumor cells with different phenotypes, which results in the growth of the primary tumor and emergence of new tumors.

CSC biomarkers
Due to the close relationship between CSCs and tumor initiation, progression, metastasis and drug resistance, the isolation of these cells from the total cancer cell population is essential for detailed studies. Distinct and specific surface biomarker phenotypes can be used to distinguish CSCs from other tumor cells and normal stem cells. Currently, the most common method used to identify CSCs is fluorescence-activated cell sorting (FACS) based on cell surface markers or intracellular molecules.
The leukemia stem cell (LSC), which specifically displays a CD34 + CD38surface marker phenotype, was the first studied and best understood type of CSC [3] . The loss of CD38 distinguishes LSCs from normal hematopoietic stem cells (HSCs), although both LSCs and HSCs are CD34 + [3] . Subsequently, more selective surface markers of LSCs have been discovered, and combinations of these markers have been used. For example, the combination of CD34 + CD38 -HLA-DR -CD71 -CD90 -CD117 -CD123 + is uniquely present on the surface of LSCs but not on normal HSCs [18] .
Regarding solid cancers, Al-Hajj et al have identified ESA + CD44 + CD24 -/ low Lineagecells as breast cancer stem cells. The authors found that compared to other cell populations, this population has a greater capacity for tumor formation in NOD/SCID mice [4] . In addition, Singh et al have found that transplantation of a very small number of CD133 + brain cancer cells can generate tumors in NOD/SCID mice, suggesting that CD133 might be an important CSC marker [19] .
Although great progress has been made in understanding CSC surface molecules, many issues still remain to be addressed. Here, we summarize partial phenotypes of CSC markers according to the cancer types (Table 1). However, we must realize that CSC markers are still not perfect; not all CSCs express the markers, and some non-CSC cancer cells may also express the markers. For this reason, the markers ATP-binding cassette ATP-binding cassette (ABC) transporters are membrane transporters that can pump various distinct and structurally unrelated small molecules (such as cytotoxic drugs and dyes) out of cells at the expense of ATP hydrolysis. Normal stem cells and cancer stem cells appear to express high levels of ABC transporters. This phenomenon could contribute to multidrug resistance (MDR) because many anti-tumor drugs can be pumped out, thereby resulting in low intracellular drug concentrations [25,30,34] . Thus, the elevated levels of ABC transporters enable cancer stem cells to resist current cancer therapies [25,35] . The well-known "shining stars" of the ABC superfamily include multidrug resistance proteins (MRPs/ ABCC), breast cancer resistance protein (BCRP/ABCG2) and P-glycoprotein (P-gp/ABCB1) [35] . High expression levels of ABC transporters in malignant stem cells can be determined by treatment of cells with Hoechst 33342 dye. Rare cells containing high levels of the ABC transporters expulse Hoechst; this fraction is designated as side population (SP) cells [35] . Accumulated data have shown that numerous cell lines and tumors contain SP cells and that this cell population possesses a greater capacity for tumorigenesis than non-SP cells.
Key signaling pathways Dysregulation of signaling pathway networks plays an important role in enabling CSCs to retain stem cell properties. As shown in Figure 1, the pathways and elements that are involved in the control of self-renewal and differentiation of cancer stem cells and normal stem cells include PI3K/ Akt, PTEN [36] , JAK/STAT, Wnt/β-catenin, hedgehog, Notch, NF-κB, Bcl-2, and others [37][38][39][40][41][42][43] . The PI3K/AKT signaling pathway is involved in numerous cancers, including acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). AKT activation is very important for cellular transformation and tumorigenesis. We have observed the role of AKT1 containing an E17K mutation in v-Abl-mediated cellular transformation [38] . The AKT1 (E17K) mutant can cause increases in the protein levels of Bcl-2 and the phosphorylation of the pro-apoptotic protein BAD, resulting in enhanced resistance to apoptosis. In addition, other members of the PI3K pathway (such as PTEN and mTOR) also function in the maintenance of LSCs. Silencing or defective mutations of PTEN have been observed in various cancers, including T-cell acute lymphoblastic leukemia, prostate cancer, melanoma, glioblastoma and endometrial carcinoma [44] .
The JAK/STAT signaling pathway is also involved in tumor initiation. Aberrations in the JAK/STAT pathway have been recognized in many cancers, especially leukemia. v-Abl is a strong non-receptor tyrosine kinase that can induce the malignant transformation of pre-B cells by affecting the JAK/STAT pathway. Pim-1 and Pim-2 kinases have been shown to be induced by v-Abl-dependent JAK/STAT signaling in pre-B cells and play important roles in cellular transformation [45] . In addition, the JAK2-V617F mutation has been shown to be a critical factor that contributes to the malignant transformation of hematopoietic cells [46] . An important mechanism for negative regulation of the JAK/STAT signaling pathway is mediated by members of the suppressor of cytokine signaling (SOCS) family. Because activation of JAK/STAT signaling is required for transformation by several oncogenes, overcoming the regulatory effects of SOCS-1 and SOCS-3 has been proposed to be necessary for tumor formation. Indeed, signaling activated by Bcr-Abl, a tyrosine kinase that causes CML, has been shown to lead to tyrosine phosphorylation of SOCS-1 and SOCS-3, thereby impairing the ability of these proteins to inhibit JAK/STAT signaling pathway activation [47] .
Nuclear factor kappa B (NF-κB), a transcription factor that regulates the expression of multiple genes, participates in numerous cellular responses to stimuli such as cytokines, microbial antigens, free radicals and ultraviolet irradiation [39] . An increasing body of evidence has shown that NF-κB affects the expression of several apoptosis-related proteins, such as Bcl-xL, Bcl-2, survivin, cellular inhibitors of apoptosis (cIAPs), TRAF and cell cycle regulatory components. Aberrant NF-κB activation causes cancer development and progression, chemoresistance, chronic inflammation and autoimmune diseases [48,49] .
The well-known Notch, Hedgehog and Wnt signaling pathways play fundamental roles in maintaining CSC populations. Notch signaling affects self-renewal and lineage-specific differentiation of normal human breast stem cells [50] . Moreover, Notch4 activity is elevated in breast CSCs, and inhibiting Notch4 activity can reduce the breast CSC population, thereby suppressing tumor initiation [42] .
Aberrant regulation of the Hedgehog pathway is associated www.chinaphar.com Chen K et al Acta Pharmacologica Sinica npg with numerous human malignancies and is a critical factor affecting the outcome of treating the disease [51] . Several agents targeting the Hedgehog pathway have shown promising preclinical results and are currently under investigation in phase I and II clinical trials [51] . The maintenance of CSC stem cell properties has been shown to be associated with the Wnt signaling pathway. Abnormal Wnt/β-catenin signaling has been identified in various malignancies, such as leukemia, colon, epidermal, breast and cutaneous carcinoma [52,53] . Human colon carcinoma, one of the best studied tumors, is caused by defective mutations in the adenomatous polyposis coli (APC) gene [54] . Defective mutations in APC result in the inappropriate stabilization of β-catenin, thus activating the Wnt cascade and inducing epithelial cell transformation [54] .

Therapies targeting cancer stem cells
Current therapeutic strategies against cancer have severe limitations that frequently lead to treatment failure. A common cause of treatment failure in multiple malignancies is resistance to chemotherapy and radiotherapy. In addition, many strategies that are not sufficiently selective against CSCs can be toxic to healthy tissues, and patients usually face the risk of recurrence and metastasis because most therapies cannot eliminate CSCs [55] . Accumulated evidence has established that CSC populations are more resistant to conventional cancer therapies than non-CSC populations. Therefore, the elimination of CSCs is crucial in treating malignant diseases [56][57][58] .
Recently, multiple novel therapeutic systems have been designed with the aim of killing CSCs and altering the microenvironment (niches) supporting these cells. Both subtle surface marker differences and alterations in signaling pathways are alluring therapeutic targets. Scientists have identified multiple potential CSC therapeutic targets, including the ABC superfamily, anti-apoptotic factors, detoxifying enzymes, DNA repair enzymes and distinct oncogenic cascades (such as the Wnt/β-catenin, hedgehog, EGFR and Notch path ways) [58,59] . Currently, some therapeutic strategies can successfully kill CSCs, while others are still under preclinical and clinical evaluation. A summary of new CSC-targeted therapeutic strategies is shown in Figure 2.
Targeting cellular surface markers Researchers often choose ligands or antibodies against tumor surface makers to enhance the specificity of therapeutic strategies. Important interest has been generated in the development of monoclonal antibodies to target CSCs. For example, CD33 is highly expressed in most AML cells. Thus, gemtuzumab ozogamicin, a humanized anti-CD33 mouse monoclonal antibody conjugated to the cytotoxic agent calicheamicin, has been developed and widely used to treat AML [60] .
Markers differentially expressed between normal stem cells and CSCs, including CD44 [61] , IL-3R [62] , and the immunoglobulin mucin TIM-3 [63] , have been utilized to specifically target leukemia stem cells in human AML. In each case, treatment with antibodies against these cell surface molecules dramati-cally decreases leukemogenicity and eradicates CSCs in mice. Antibodies against CD47, which is expressed at much higher levels in ALL than in normal cells, may also effectively kill leukemia stem cells [64] . In treating MCF-7 breast cancer, an anti-CD44 antibody-conjugated gold nanorod has been used to target and photo-ablate CD44 + cells, which display significant cancer stem cell characteristics. Using this approach, targeted cells absorb near infrared light, which results in increased local temperature at the designated location [65] .
An anti-CD90 antibody has also been reported to protect bone marrow-derived multipotent stromal cells from differentiation into chondro-, osteogenic, or adipo-lineages [66] . In addition, CD133 is highly expressed in many types of CSCs, including lung cancer, breast cancer, and glioma. Patients with high levels of CD133 show poor clinical outcomes [67] . For example, increased CD133 mRNA expression correlates with a high risk of death in colon, prostate and head and neck cancer [68] . Thus, therapy against CD133 might represent a promising strategy for cancer treatment. CD133 + cells in glioblastoma (GBM) display cancer stem cell-like properties [69] . Interestingly, a recent study has shown that treatment with carbon nanotubes con- Figure 2. Therapies targeting cancer stem cells. Numerous therapies aiming at eradicating cancer stem cells have been developed during these years. Four different areas could recapitulate the current popular ideas mostly. By selectively targeting surface markers of CSCs (red area), more accurate and less side effects could be achieved. With the help of modern molecular biology techniques, more and more crucial signal elements and pathways have been unearthed (green area). By intervening aberrant pathways, specific characteristics of CSCs are suppressed and promising outcome has been reported. Molecular drugs inhibiting ABC cassette (purple area) have reached the third generation (tariquidar) and some of them are undergoing clinical trials. In addition, tumor microenvironment nursing CSCs also draws much attention (blue area). Either cutting off the growth of blood vessel or exploiting the special PH environment, has showed alluring prospects.
www.nature.com/aps Chen K et al Acta Pharmacologica Sinica npg jugated to an anti-CD133 monoclonal antibody followed by irradiation with near infrared laser light can selectively target CD133 + GBM cells, and the photothermolysis caused by the nanotubes can kill the targeted cells [69] . In addition, disruption of CD133 expression by short hairpin RNA in human glioblastoma neurospheres has been shown to impair the self-renewal and tumorigenic capacity of the neurosphere cells [70] .
Targeting ATP-driven efflux transporters Antitumor drug efflux caused by ATP-driven pumps is the primary reason for chemoresistance [55,71,72] . Investigators have designed numerous methods to evade, neutralize or even exploit drug efflux pumps to overcome drug resistance. Several pharmacological agents that can interact with ABC transporters have been developed to inhibit multidrug resistance (MDR) [73,74] . The first P-gp efflux pump inhibitor to be identified was verapamil, which is often used in SP analysis to block the exclusion of Hoechst dye [75] . Simultaneous treatment with verapamil and antitumor drugs, such as Dox, paclitaxel or vincristine, has displayed promising therapeutic effects.
Khdair et al have delivered methylene blue (a type of P-gp inhibitor) and Dox simultaneously into BALB/c mice bearing syngeneic JC adenocarcinoma tumors. Markedly increased accumulation of drugs within the lesion, enhanced tumor cell apoptosis, suppressed cancer cell proliferation, impaired tumor growth and significantly improved animal survival were observed [76] .
Some investigations have identified a number of novel ABC transporter inhibitors that suppress both P-gp and MRP1, such as MS-209, VX-710, and tariquidar [73,77,78] . Recent trials with MS-209 have shown promising results in overcoming drug resistance in breast cancer [77] and other solid cancers (ClinicalTrials.gov Identifier: NCT00004886). Tariquidar is another inspiring inhibitor. The combination of tariquidar and docetaxel is under investigation (sponsored by the National Cancer Institute) for the treatment of recurrent or metastatic ovarian, cervical, lung and kidney malignancies (ClinicalTrials.gov identifier: NCT00072202). Similarly, the combination of tariquidar with Dox, etoposide, mitotane and vincristine is being tested in patients with primary, recurrent, and metastatic adrenocortical cancer (Clinical-Trials.gov identifier: NCT00073996) [73] .
An alternative strategy targeting ABC transporters involves regulating the protein expression levels of these transporters. Sims-Mourtada et al have indicated that Hedgehog signaling can regulate the expression of MDR1 and ABCG2. The expression levels of ABCG2 and MDR1 are downregulated upon treatment of PC3 cells with cyclopamine (a SMO signaling element inhibitor), and targeted knockdown of ABCG2 and MDR1 expression by siRNA partially reverses chemoresistance [79] .
Targeting key signaling cascades Active anti-apoptotic pathways and parallel inactive proapoptotic pathways are also hot spots attracting researchers.
Monoclonal antibodies targeting Notch signaling have shown alluring prospects [80,81] . Inhibition of Notch1 can significantly reduce the CD44 + CD24 −/low subpopulation and lower the incidence of brain metastases from a breast cancer cell line [82] .
High levels of β-catenin have been described to correlate with CSC tumorigenicity in colon cancer [83] . Several pharmaceuticals are under investigation as inhibitors of Wnt signaling [84,85] . Monoclonal antibodies against the Wnt cascade have been tested with promising anti-tumor activity [86] .
Small-molecule Hedgehog antagonists have shown alluring potential to inhibit systemic metastases in mice bearing orthotopic xenografts from human pancreatic cancer cell lines. A significant reduction in the population of ALDH-positive cells (the tumor-initiating population in pancreatic cancer) has also been observed [87] . Several groups have exploited cyclopamine, a SMO signaling element inhibitor, to inhibit the hedgehog cascade, and cyclopamine has been shown to inhibit the growth, invasion and metastasis of breast, prostatic, pancreatic and brain malignancies both in vitro and in vivo [88][89][90][91] . Cyclopamine can act synergistically with gemcitabine to reduce the population of ALDH high pancreatic CSCs [92] . Similarly, Clement et al have demonstrated that the combination of cyclopamine and temozolomide (TMZ) can also reduce the cell mass of glioma CSCs in vivo [93] .
Several groups have found that inhibiting NF-κB can greatly suppress chemoresistance, and inhibitors targeting NF-κB can mediate antitumor responses and enhance the sensitivity of tumor cells to anticancer drugs. Simultaneous delivery of DOX and the NF-κB inhibitor PDTC successfully overcomes multidrug resistance [94] . Ganta and Amiji have also observed enhanced cell apoptosis upon treatment with paclitaxel and curcumin, another NF-κB inhibitor [95] . Curcumin can dampen the protein levels of MDR1 [96] and activate apoptosis pathways, hence impairing the capacity for multidrug resistance.
Ceramide, a secondary lipid messenger implicated in the regulation of cell proliferation, apoptosis and differentiation, has drawn much attention as a pro-apoptotic molecule that can activate apoptotic pathways via receptor independent mechanisms [97] . Devalapally et al have increased the level of ceramide in drug resistant cancer cells to lower the threshold for apoptotic signaling. Tamoxifen can inhibit the activity of ceramide-metabolizing enzymes and thus increase intracellular ceramide levels. Drug resistance in P-gp-overexpressing SKOV3(TR) cells is significantly suppressed upon simultaneous delivery of paclitaxel and tamoxifen [97] .
In addition, IL-4 can protect the tumorigenic CD133 + CSCs in human colon carcinoma from apoptosis. Thus, an anti-IL-4 antibody or IL-4Ra antagonist can cause CSCs to become apoptotic and markedly sensitize CSCs to chemotherapeutic drugs [41] .

Targeting the tumor microenvironment
The tumor microenvironment can create a niche to nurse and protect CSCs from drug-induced apoptosis. For example, most mature B-cell malignancies are incurable. Compelling evidence has suggested that accessory stromal cells in the www.chinaphar.com Chen K et al Acta Pharmacologica Sinica npg tissue microenvironments of bone marrow and secondary lymphoid organs favor disease progression by promoting malignant B-cell growth, proliferation and drug resistance [98] . One example is the interaction between stromal cell-derived factor-1 (SDF-1/CXCL12), secreted by bone marrow stromal cells, and its receptor CXCR4 [40] . CXCR4 can guide leukemia cell trafficking and homing to the bone marrow microenvironment, where secreted CXCL12 ensures that leukemia cells closely contact the marrow stromal cells, consequently activating cell growth and chemoresistance signals. CXCR4 antagonists, such as Plerixafor (AMD3100) and T14003 analogs, can damage adhesive tumor-stroma interactions and induce leukemia cell mobilization away from the bone marrow stromal microenvironment, causing the cells to become more vulnerable to cytotoxic drugs. The novel approach of targeting the CXCR4-CXCL12 axis is being explored in clinical trials for leukemia [40,98] .
Tumor angiogenesis has also been reported to be related to CSC survival and drug resistance. VEGF has been recognized to correlate with microvasculature formation and tumor growth. Targeting VEGF with bevacizumab leads to the normalization of tumor vasculature, resulting in a disruption of the CSC niche. Previous studies have demonstrated that treatment of mouse glioblastoma with bevacizumab causes a dramatic reduction in the number of glioblastoma stem cells [99] . Calabrese et al have treated mice bearing U87 glioma cell xenografts with bevacizumab and observed decreased microvasculature density and tumor growth. The authors also observed a reduction in the number of CD133 + /nestin + tumorinitiating cells [100] . The combination of the VEGFR2 antibody DC101 and the cytotoxic agent cyclophosphamide is also more effective against C6 glioma xenografts in vivo than individual agents alone [101] .
Weakly acidic pH is a well-known characteristic of the microenvironment of solid tumors. TAT peptide is an arginine-rich peptide (YGRKKRRQRRR) that currently serves to rapidly transport various types of attached molecules, such as small molecule drugs, into mammalian cells both in vitro and in vivo. Lee et al have introduced TAT to a pH-sensitive cargo that is also conjugated to biotin. This cargo exposes biotin on its surface under slightly acidic pH values (6.5<pH<7.2). Thus, the pH environment together with exposed biotin can greatly increase the specificity of TAT. The latter quickly elevates the concentration of Dox in the cytosol. These processes have been reported to significantly increase the potency of Dox. This novel cargo has been evaluated in tumor xenografts of non-small cell lung cancer A549 cells, A2780/AD-resistant breast cancer MCF-7 cells and nasopharyngeal carcinoma KB cells; an obvious reduction was observed in the size of all tumors [102] .

Conclusions
In summary, compelling evidence has shown that CSCs indeed exist in various malignancies and display capacities for self-renewal and differentiation that are critical for tumor initiation, progression, metastasis and recurrence. Currently, the identification and isolation of CSCs are primarily based on CSC surface markers. However, CSC markers are not universal, and CSC populations have repeatedly been refined due to the identification of new markers. Thus, to what extent will a suitable number of makers be identified to define the real population of CSCs? Would these biomarkers change during cancer progression? These questions still remain to be addressed.
Designing novel approaches to target CSCs has received much attention over the past several years. Increasing evidence has suggested that a comprehensive strategy might improve cancer treatments. For example, a nanoparticle conjugated to the following four crucial elements has been designed: 1) a molecule (ie, ligand) targeting a specific CSC; 2) a cytotoxic anticancer drug to eliminate CSCs; 3) a chemosensitizer to overcome drug resistance (such as an ABC transporter inhibitor); and 4) an imaging agent to facilitate tumor diagnostics. Such a combination would exert the anti-tumor effect more specifically and powerfully with fewer side-effects. Furthermore, this approach would enable accurate identification of the localization of the primary tumor and its metastases. The rise of the cancer stem cell hypothesis broadens our horizons and provides a new approach to eradicate malignancies. However, the identification of strategies that exploit the unique characteristics of CSCs requires further study and the cooperation of multidisciplinary areas.