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Drosophila Females Undergo Genome Expansion after Interspecific Hybridization

Genome size (or C-value) can present a wide range of values among eukaryotes. This variation has been attributed to differences in the amplification and deletion of different noncoding repetitive sequences, particularly transposable elements (TEs). TEs can be activated under different stress conditions such as interspecific hybridization events, as described for several species of animals and plants. These massive transposition episodes can lead to considerable genome expansions that could ultimately be involved in hybrid speciation processes. Here, we describe the effects of hybridization and introgression on genome size of Drosophila hybrids. We measured the genome size of two close Drosophila species, Drosophila buzzatii and Drosophila koepferae, their F1 offspring and the offspring from three generations of backcrossed hybrids; where mobilization of up to 28 different TEs was previously detected. We show that hybrid females indeed present a genome expansion, especially in the first backcross, which could likely be explained by transposition events. Hybrid males, which exhibit more variable C-values among individuals of the same generation, do not present an increased genome size. Thus, we demonstrate that the impact of hybridization on genome size can be detected through flow cytometry and is sex-dependent.

INTRODUCTION
Genome size, also known as C-value, is the measure of DNA mass per haploid nucleus (Gregory 2005b) and represents a crucial feature for the understanding of genome evolution and speciation (Kraaijeveld 2010). Although this value is constant within individuals, eukaryotic species present a wide variation in genome size, reaching differences higher than 600,000-fold (Gregory 2005a). The lack of correlation between organisms' genome size and their number of genes or their complexity was called the "C-value paradox," an issue that was cleared up by the finding that genes are not the only (nor the major) components of genomes. It is now known that a large fraction of the genome of most eukaryotic organisms is noncoding repetitive DNA, including transposable elements (TEs), pseudogenes, introns, and satellites (Gregory 2005a). Together with polyploidization, transposition is considered to be one of the major forces of eukaryotic genome expansion (Kidwell 2002): for instance, the maize genome doubled its size during the last few million years after a series of transposition bursts (SanMiguel et al. 1996). In the Drosophila genus, some studies have demonstrated that TE amount can account for genome size variation between species (Boulesteix et al. 2006), as well as between populations of the same species (Vieira et al. 2002).
Although TE mobilization rates are usually low, spontaneous transposition bursts have been reported, often linked to different stressful conditions (reviewed in García Guerreiro 2012). Interestingly, some of these bursts seem to share timing with species radiation episodes (Rebollo et al. 2010).
The merge of two different genomes during interspecific hybridization events can be considered a genomic stress condition, which has been shown to lead to transposition bursts in several species. For example, different macropodid hybrids present amplified centromeres due to the presence of TE-related sequences (O'Neill et al. 1998;Metcalfe et al. 2007), and retrotransposon proliferation has also been described in three sunflower species of hybrid origin (Ungerer et al. 2006).
In Drosophila, the first evidence of hybrid TE mobilization was the detection of a new insertion of the pDv111 element in Drosophila virilis-Drosophila littoralis hybrids by in situ hybridization (Evgen'ev et al. 1982). In the same way, an increase of transposition of the retrotransposon Osvaldo was reported in hybrids between Drosophila buzzatii and Drosophila koepferae (Labrador et al. 1999). More recently, a genome-wide study using AFLP markers in these same hybrids demonstrated that not only Osvaldo but at least 28 different TEs were mobilized (Vela et al. 2014), suggesting that transposition in D. buzzatii-D. koepferae hybrids is a widespread phenomenon. Other studies at a transcription level support the hypothesis of a TE derepression in hybrids between Drosophila species (Kelleher et al. 2012;Carnelossi et al. 2014;García Guerreiro 2015), as well as in hybrid lake whitefishes (Dion-Cô té et al. 2014) and sunflowers .
Massive bursts of transposition can cause drastic changes in genome size and composition. For instance, three hybridderived sunflower species present genome sizes 50% larger than parental species (Baack et al. 2005). This study shows that interspecific hybridization is a source of evolutionary novelties that may be at the origin of new species by the means of TE activation (reviewed in Fontdevila 2005;Rebollo et al. 2010). However, synthetic F 1 and F 6 hybrids between the same sunflower parental species do not present a genome increase, and neither do plants from hybrid-zone populations (Baack et al. 2005;Kawakami et al. 2011). These last results show that genome expansion is not a shared feature of all interspecific hybrids, which concurs with studies in other plants, such as oil palm, sea buckthorns, and grasses, where hybrids presented intermediate genome sizes between parental species (Mahelka et al. 2005;Zhou et al. 2010;Camillo et al. 2014).
In animals, despite the few studies describing TE activation in hybrids (O'Neill et al. 1998;Labrador et al. 1999;Metcalfe et al. 2007;Vela et al. 2014), information about the effect of hybridization on hybrid genome size is scarce. D. buzzatii and D. koepferae are two cactophilic species that only produce hybrid offspring when crossing D. buzzatii males with D. koepferae females-the reciprocal cross does not produce adult offspring (Marin et al. 1993). As previously mentioned, mobilization of different TEs in hybrids between these species has been reported by in situ hybridization, AFLPs and transposon display techniques (Labrador et al. 1999;Vela et al. 2011Vela et al. , 2014. We have estimated the genome size of these two parental species and their F 1 hybrids, as well as three subsequent generations of backcrossed hybrids ( fig. 1). Thus, the present work aims to analyze the impact of interspecific hybridization, at different stages of genomic introgression, on genome size of male and female Drosophila hybrids.

Drosophila Stocks and Crosses
Six interspecific crosses were performed between ten D. buzzatii males (Bu28 strain) and ten D. koepferae females (Ko2 strain). Both strains are inbred lines originated by natural populations collected, respectively, in Bolivia and Argentina (Morá n and Fontdevila 2014). Each cross was followed by three generations of backcrossing of ten hybrid females with ten D. buzzatii males. All stocks and crosses were reared at 25 C in a standard Drosophila medium.

Genome Size Estimation
Genome size of D. buzzatii, D. koepferae, and their hybrids was estimated for males and females separately using flow cytometry technique. Nuclei were extracted from three heads of exactly 4 days-old flies, using D. virilis as internal control standard. Heads were homogenized in Galbraith buffer (30 mM trisodium citrate, 10 À4 triton X-100, 2 mg/ml RNAse A, 20 mM MOPS, 21.3 mM MgCl 2 ) with 0.1 mg/ml propidium iodide (pH 7.2). After two filtering steps through 140 and 30-micron nylon meshes, samples were analyzed on a FACSCanto II flow cytometer fitted with an argon laser (488 nm wavelength). The relative fluorescence intensity between our flies and D. virilis, whose genome size estimate is 0.34 pg (Gregory and Johnston 2008), was determined. We performed 5-6 biological replicates for parental samples and 8-10 for hybrids (supplementary table  S3, Supplementary Material online).

Statistical Analyses
Comparisons between parental species genome sizes were performed using the nonparametric Wilcoxon rank sum test (Mann and Whitney 1947), while hybrid genome size estimates were compared to a single theoretical mean (specific to each generation) with the Wilcoxon signed-rank test (Wilcoxon 1945). The single theoretical value specific to each generation was calculated for males and females separately, as follows: where f is the D. buzzatii (f bu ) or D. koepferae (f ko ) mean genome fraction of each generation (for example, for BC1, f bu ¼ 0:75 and f ko ¼ 0:25) and GS is the mean genome size of D. buzzatii (GS bu ) or D. koepferae (GS ko ).

AFLP Genotyping
AFLP technique was suitable for our study because it did not require prior information on our species sequences (D.
koepferae available sequences are scarce) and had previously been used in our species and their hybrids (Morá n and Fontdevila 2014; Vela et al. 2014). Markers were obtained following the protocol described in Vela et al. 2011, from six hybrid crosses used in a former study (Vela et al. 2014). Contrary to the previous study, where instability markers were checked, we here identified D. koepferae-specific markers for ten primer combinations (supplementary table S2

Results and discussion
As a first goal, we have determined both D. buzzatii and D. koepferae parental genome sizes to assess differences between them, and also between males and females. Our results show that D. buzzatii presents a mean C-value of 176.28 Mb for females and 169.07 Mb for males ( fig. 2A). These values are significantly higher (P = 0.022, supplementary table S1A, Supplementary Material online) than the 146-153 Mb previously reported by other authors (Guillé n et al. 2015). It is known that differences in the estimated values can depend on the technique used (flow cytometry vs. densitometry), the analyzed tissues (heads vs. testes) or even on fly rearing conditions (Nardon et al. 2003). Furthermore, it is important to note that the genome size reference used in the former study (Guillé n et al. 2015) was the Drosophila mojavensis genome assembly size, which could likely suppose an a priori underestimation due to assembling issues. Indeed, most of the repeated sequences are not assembled and we know they can contribute to genome size variation. On the other hand, intraspecific variation in Drosophila genome size among different strains or populations has been reported in several studies (Vieira et al. 2002;Bosco et al. 2007;Gregory and Johnston 2008;Ellis et al. 2014). These differences have been attributed to changes in TE (Vieira et al. 2002) and satellite DNA amounts (Bosco et al. 2007), and seem to be correlated with several life history traits and metabolism genes expression (Ellis et al. 2014).
In this study, we globally observe that parental females have significantly larger genomes than males (P = 1.48E-06, supplementary table S1A Supplementary Material online), with differences of approximately 7 Mb for both species ( fig. 2A). Similar results have been described in Drosophila mauritania or Drosophila hydei (Girard and Hannon 2008), but Drosophila melanogaster presents equivalent genome sizes for both sexes (Vieira et al. 2002) and Drosophila simulans males exhibit larger genomes than females (Vieira et al. 2002). However, different results have been found in other strains of the latter two species (Gregory and Johnston 2008), indicating that genome size differences between males and females are strain-specific and likely depend on specific increases of repetitive DNA in the Y chromosome heterochromatin (Vieira et al. 2002). In our species, we expect females to have a higher genome size than males because X chromosome is known to be longer than Y (Wasserman 1962;Fontdevila et al. 1988). Interestingly, the standard errors observed within replicates are &2-fold higher in males than in females, showing that males present greater genome size variability (supplementary table S1, Supplementary Material online). The dynamic gene content of the Y chromosome, which also contains a high amount of repetitive sequences, might account for this diversity (Bernardo Carvalho et al. 2009).
Differences between species are significant for females (P = 0.015, supplementary table S1A, Supplementary Material online), with D. koepferae genome about 3 Mb larger than D. buzzatii ( fig. 2A). No significant difference was observed in males (P = 0.126, supplementary table S1A, Supplementary Material online), which is probably due to the lower genome size and the higher variability found in male samples.
According to our null hypothesis, the genome size of hybrids (F 1 and backcrosses) would present intermediate values between parental species and would be proportional to the D. buzzatii/D. koepferae genome fractions at each generation ( fig. 1). Thus, we have compared the C-values of each hybrid generation to a theoretical weighted mean, reflecting the expected mean D. buzzatii introgression percentage in the hybrid genomes, assuming independent assortment of chromosomes during meiosis (see Materials and Methods). The accuracy of this assumption has been tested through AFLP genotyping of hybrids and parents: we have used 70 AFLP markers specific to D. koepferae and assessed which proportion of these markers is transmitted to hybrid progeny (see below). A first interspecific massal cross of ten D. koepferae females with ten D. buzzatii males was followed by three subsequent backcrosses of ten hybrid females with ten D. buzzatii males. The D. buzzatii expected mean genome fraction of each generation is presented in parentheses.
In females, we show that the mean genome size of the four hybrid generations is higher than the theoretical value ( fig. 2B), with statistically significant differences for the three backcrosses (supplementary table S1B, Supplementary Material online). The most striking results occur in the first backcross (BC1): the mean C-value (180.37 Mb) increases compared with the F 1 generation (178.23 Mb), and is also higher than in both parental species (176.28 and 179.08 Mb). These results are concordant with the transposition-related instability observed previously in our hybrids, where new insertions of 28 different TEs, including retrotransposons and DNA transposons, were detected in the three backcrosses (Vela et al. 2014). In the case of F 1 , the vast majority of the detected instability markers were not transmitted to BC1, showing that the putative transposition events of F 1 take place after meiosis (Vela 2012), which is also coherent with our results: somatic transpositions are not expected to cause a genome size increase. TE activation in hybrids seems to be caused by the failure of epigenetic repression mechanisms (Michalak 2009), such as histone methylation or small RNA biogenesis. In Drosophila ovaries, TEs are mainly regulated by piRNAs, a kind of small RNAs associated to Piwi proteins. Differences in piRNA pools between parental species, or incompatibilities between their piRNA pathway effector proteins, might lead to a TE silencing failure in hybrids. If a TE derepression took place in F 1 ovaries at a transcriptional level, as shown for D. simulans-D. melanogaster hybrids (Kelleher et al. 2012), we would expect to detect new insertions in the following generations. Thus, new TE insertions could likely be responsible for the genome size increase observed after F 1 .
It is worth noting that other phenomena could also account for the observed genome expansion, such as the amplification of satellites or other noncoding repetitive sequences (Bosco et al. 2007), which are responsible for the large Drosophila orena genome (Boulesteix et al. 2006). On the contrary, polyploidization can be discarded, since early studies of D. buzzatii-D. koepferae hybrids, based in in situ hybridization (Labrador et al. 1999), never reported a case of hybrid abnormal karyotype due to genome duplication.
Finally, a transmission bias favoring the larger parental genome, D. koepferae, could also be consistent with our results (e.g., due to reduced recombination or differential gamete viability). In order to test this hypothesis, we have determined the inheritance of 70 D. koepferae-specific AFLP markers in F 1 and BC1 hybrids from six different crosses. Our results, summarized in supplementary table S2, Supplementary Material online, show that almost 100% (92.9-97.1%) of the studied D. koepferae-specific markers are found in F 1 , as expected: all F 1 individuals have an entire haploid copy of the D. koepferae genome. In the BC1, between 11.8% and 72.9% of the markers are found per individual (supplementary table S2, Supplementary Material online). This variability was also predictable, because inheritance of D. koepferae markers depends on the chromosomal assortment and recombination events occurring in each F 1 gamete. Thus, it is not surprising that BC1 and BC2 hybrids present higher standard errors on genome size measurements than parental species (supplementary table S1B, Supplementary Material online and fig 2B). The average proportion of D. koepferae markers found in BC1, 32.4% (95% confidence interval: 11.6-53.2%), is lower than the expected mean of 50%, which suggests that either the transmission of the smallest parental genome (D. buzzatii) is favored in BC1 hybrids, or there is not any transmission bias. It is also worth noting than even considering the most extremely biased case (P = (½) 5 = 0.03), in which 1) there is no recombination between D. buzzatii and D. koepferae chromosomes and 2) all individuals inherit all five D. koepferae chromosomes from their hybrid mothers; the D. koepferae genome fraction in backcrossed hybrids would be of 50% (as in F 1 ). Assuming these improbable particulars, genome size estimates remain significantly higher than the expected for BC1 (Wilcoxon signed-rank test V = 49, P = 0.027).
Despite the genome size is higher than expected in all backcrosses ( fig. 2B), its value actually decreases through generations after BC1 ( fig. 2B). In rice (Oryza sativa), an important increase of Tos17 and RCS1 retrotransposons copy number was observed after introgression with Zizania latifolia, but no additional insertions were detected after a few generations (Liu and Wendel 2000), meaning that TE mobilization was by then controlled. Thereby, we can suppose that after a few generations of introgression, the preponderance of one of the parental genomes mitigates incompatibilities and palliates the hybridization effects. In this way, we can hypothesize that a greater transposition control in our hybrids would take place after BC1, which according to previous studies is true for the transposon Galileo, but not for Helena (high transposition rates observed also in BC2) and Osvaldo (higher transposition rates in BC3) (Vela et al. 2014). However, these elements represent only a small subset of these species' TEs and may not be representative of the whole set behavior.
The simple backcrossing with D. buzzatii (species with smallest genome) could by itself lead to the observed genome size decrease after BC1, but active mechanisms involving genome reduction might also be involved, especially those implicated in TE control. For instance, it is known that internal and complete deletions of TE copies can act as a prevention mechanism against genome invasions (Petrov and Hartl 1998;Liu and Wendel 2000;Senerchia et al. 2015), the latter being guided by the presence of multiple TE copies through recombination events.
The observed genome increase in hybrid females could also be a technical artefact due to changes in chromatin topology. In hybrids, the failure to maintain chromatin integrity could improve the accessibility of DNA to fluorochromes (Nardon et al. 2003), resulting in an increase of genome size estimates. However, this hypothesis can be discarded because the lowest levels of chromatin compaction are expected in F 1 , whereas the highest genome size measures belong to BC1.
Regarding our hybrid males, it is worth mentioning that they are all sterile until BC3, when fertility is recovered for some individuals (Morá n and Fontdevila 2014). Here, we show that all hybrid generations present intermediate genome size values between D. buzzatii and D. koepferae ( fig. 2C and supplementary table S1B, Supplementary Material online). Although the mean C-value of each generation is higher than the theoretical, differences are not significant (supplementary table S1B, Supplementary Material online), meaning that the impact of hybridization and introgression on genome size is negligible in males. This seems contradictory with the fact that new TE insertions in our hybrids were also detected in males (Vela et al. 2014), where Osvaldo transcription rates were higher than in parents (García Guerreiro 2015). However, these male transposition events were thought to be partly somatic (Vela et al. 2014), and thus would not necessarily lead to a genome expansion. Furthermore, other transposons, such as Helena, seem to be repressed in hybrid males (Romero-Soriano and García Guerreiro 2016). This shows that TE regulation patterns differ between sexes and depend on the studied TEs, as proposed in a recent study (Senti et al. 2015). Indeed, the biogenesis of piRNAs has been shown to differ between males and females (Nagao et al. 2010;Siomi et al. 2010). Although we cannot rule out the involvement of particular TEs in the hybrid male sterile phenotype, our results suggest that, unlike hybrid females, males do not present a massive TE amplification.

Conclusions
We have shown that the increased transpositional activity previously reported in D. buzzatii-D. koepferae hybrids has an impact on hybrid female genome size. For the first time, an actual genome size increase due to interspecific hybridization has been described in animals. This allows us to validate flow cytometry as a technique to detect changes in C-value of Drosophila hybrids, probably due to transposition events. In males, the effects of hybridization are not significant, but we must note that changes in their genome size would lack direct evolutionary consequences, since they are all sterile until some individuals recover their fertility in BC3.


s2fieldsofstudy: Biology Medicine

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Neonatal disease environment limits the efficacy of retinal transplantation in the LCA8 mouse model

Background Mutations of Crb1 gene cause irreversible and incurable visual impairment in humans. This study aims to use an LCA8-like mouse model to identify host-mediated responses that might interfere with survival, retinal integration and differentiation of grafted cells during neonatal cell therapy. Methods Mixed retinal donor cells (1 ~ 2 × 104) isolated from neural retinas of neonatal eGFP transgenic mice were injected into the subretinal space of LCA8-like model neonatal mice. Markers of specific cell types were used to analyze microglial attraction, CSPG induction and retinal cell differentiation. The positions of host retinal cells were traced according to their laminar location during disease progression to look for host cell rearrangements that might inhibit retinal integration of the transplanted cells. Results Transplanted retinal cells showed poor survival and attracted microglial cells, but CSPG was not greatly induced. Retinas of the LCA8 model hosts underwent significant cellular rearrangement, including rosette formation and apical displacement of inner retinal cells. Conclusions Local disease environment, particularly host immune responses to injected cells and formation of a physical barrier caused by apical migration of host retinal cells upon disruption of outer limiting membrane, may impose two major barriers in LCAs cell transplantation therapy.

Background
Photoreceptor degeneration leads to irreversible blindness in related retinal degenerative diseases, including Retinitis Pigmentosa (RP) and Leber Congenital Amaurosis (LCA) [1,2]. The early-onset retinal degeneration (RD) responsible for LCA, the severe disease leading to childhood blindness in from 1 in 30,000 to 1 in 81,000 subjects, is caused by mutation in approximately 22 genes implicated in photoreceptor function, structure and development [1,3,4]. Although no effective treatment or cure is currently available, recent advances in cell transplantation have greatly improved the survival, integration and differentiation of donor cells as well as functional recovery in animal models [5].
Two major barriers to cell-based ocular therapy are intrinsic limitations of donor cells that affect survival, migration and differentiation, and adverse host-mediated reactions to subretinal injection [6][7][8]. Host-specific limitations include the physical barrier to transplanted cell migration imposed by the outer limiting membrane (OLM, also known as an external limiting membrane) located at the apical side of the outer nuclear layer (ONL). Although OLM is formed at the junctions between rod photoreceptors and Muller glial cells by junction proteins and junction -associated proteins, including Crumbs polarity complex proteins [9][10][11], the precursor of OLM is detected in early retina before these cells have been generated. Therefore, it is plausible that junctions between retinal progenitors are essential to maintain embryonic and neonatal retinal integrity during development. The local immune reaction also affects the survival of transplanted cells. In developing and mature retina, microglia (MG) control immune surveillance and homeostasis [12]. Pathogenic activation of MG is correlated with the progression of RD [13][14][15] and pharmacological immunosuppression, including of MG activation, is protective in the RD model [16] and enhances the survival of donor cells [17]. In general, host responses to degenerating retinal cells are highly variable and diseasespecific [6,[18][19][20].
Human LCA type 8 (LCA8) is caused by Crb1 gene mutation [21,22]. The most striking phenotype among related LCA8 diseases is early-onset retinal laminar disorganization triggered by the loss of cell to cell adhesion, presumably between progenitor cells, at the apical surface of the embryonic retina. In late-onset RP12 similar Crb1 genetic defects are thought to disrupt photoreceptor to Muller glial adhesion at the apical surface of the mature retina. A partial break in OLM, a major barrier to cell migration [9,23], appears to enable efficient integration of transplanted cells into host retina in Crb1 rd8/rd8 retinas [6]. Partial chemical breakdown of OLM has also been reported to facilitate retinal integration [8]. However, LCA8-like mouse models with embryonic onset, such as Crumbs 1 and/or 2 (Crb1 and/or 2) mutant mice and conditional knock out (CKO) of protein associated with lin seven-1 (Pals1, also known as MPP5), an interacting protein with Crumbs homologs, exhibit robust cellular rearrangement during and after embryonic development following disruption of cell adhesion [24][25][26][27]. In Pals1 CKO retinas, apical localization of Crumb homologs, Par3 and β-catenin is severely disturbed during and after retinal development, and embryonic retinal cells and photoreceptor cells rearrange into half-rosettes or rosettes. Retinal cells, especially inner nuclear layer (INL) and ganglion cell layer (GCL) cells, are also apically displaced. These apically displaced cells include ganglion and amacrine cells, and Muller glia cell bodies, which are normally located centrally. All of these displacements suggest that apico-basal tissue polarity is lost due to impaired cell-cell attachment. The extensive structural defects cause ERG responses of Pals1deficient eyes to be severely reduced or virtually absent. Laminar disorganization during Pals1 CKO pathogenesis is likely to provide an adverse environment for cellbased therapy, but this notion has not been evaluated.
This study uses Pals1 CKO as a model to identify components of the neonatal retinal environment that adversely affect cell transplantation in degenerating retinas undergoing laminar disorganization. The investigation identified two major host responses that predominantly inhibited the survival and integration of transplanted cells: the MG-mediated immune response and retinal rearrangement that opposed migration into the retina.

Animals and preparation of retinal cells
All animal procedures discussed in this manuscript for the ethical treatment of animals including handling, housing, surgeries, post-surgical monitoring, anesthesia and euthanasia were approved by the Institutional Animal Care and Use Committee at Temple University. eGFP (+) mice (C57BL/6-Tg(CAG-EGFP)10sb/J, The Jackson Laboratory) were genotyped under a fluorescent microscope. Pals1 f/f allele and Rx-Cre line were previously described [26]. PCR-based genotyping was performed using the primers as described. Pals1 CKO (PR; Pals1 f/f ; Rx-Cre), heterozygote (Pals1 f/+ ; Rx-Cre) and wild type (WT) control littermates (mice not carrying Rx-Cre) were typically obtained from the crosses of heterozygotes and used for the assays. Swiss Webster (SW) mice potentially containing rd 1 allele (The Jackson Laboratory) were used as additional hosts for cell transplantation experiments.
Subretinal injection via transcleral route eGFP (+) retinas were dissected from P0-P5 eGFP (C57BL/6-Tg(ACTB-EGFP)1Osb/J) strain (The Jackson Laboratory). Whole retinal cells were dissociated after 5 min trypsin incubation at 37 o C followed by trituration, and diluted to a final concentration of~2 × 10 4 cells/μl. One μl cell suspension containing approximately 1~2 × 10 4 cells and fast green dye (0.1 %) was injected into the subretinal space of the host at neonatal stages between P2 and P6 using a capillary needle via the transcleral route [28]. Retinas were mainly analyzed between P21 (n > 3) and P35 (n > 5). For SW, retinas were analyzed at P21 (n = 5). Due to substantial variation in the size of eGFP (+) grafts, which probably resulted from methodological difficulties associated with neonatal subretinal injection, and technical challenges determining total cell numbers in grafts, this study relied on qualitative, not quantitative, assessments of grafted cells and host responses.
Confocal imaging of flat-mount retinas, immunofluorescence assays of sections and 3D reconstruction of stacked images Eyes showing green fluorescence were prescreened under a fluorescent microscope after enucleation. For flat-mount analysis, eGFP (+) retinas were fixed in 4 % paraformaldehyde for 2 h. After removing overlying retinal pigment epithelium (RPE), choroid and sclera, retinas were dissected out from the lens. Five corners of the retinas were cut with a spring scissors to flatten the retina, which was subjected to permeabilization (0.5 % dimethyl sulfoxide (DMSO), 5 % Triton in phosphate buffered saline) and blocking (3 % goat serum, 0.5 % DMSO, 5 % Triton in tris-buffered saline). Primary antibodies in blocking buffer were incubated with retinas for 3 days, and then incubated with secondary antibody overnight. Retinas stained with Hoechst 33258 were mounted with Fluoromount-G (SouthernBiotech.) on the slide (with photoreceptor side up) for confocal imaging (TCS SP8, Leica Microsystems GmbH, Germany). Stacks of images covering approximately half of the retinal thickness (2 μm steps) were typically taken from the photoreceptor side. Imaris (Bitplane) was used for 3D reconstruction of the stacks of confocal images. For section antibody staining, eGFP (+) eyeballs were fixed in 4 % paraformaldehyde and dehydrated through an ethanol series. Seven-micron thick paraffin sections were cut, and transplanted cells were detected by anti-GFP staining.

Transplantation of eGFP (+) retinal cells into neonatal Pals CKO retinas
LCA8 is characterized by early-onset visual impairment associated with disruption of OLM and subsequent retinal folding and rosette formation. In order to evaluate survival of transplanted cells in the LCA8 disease environment, approximately 20,000 dissociated enhanced green fluorescent protein (eGFP) (+) retinal cells from neonatal C57BL/6-Tg(CAG-EGFP)10sb/J) pups (P0 -P5) were subretinally injected into neonatal Pals1 CKO retinas. This model mimics the critical pathophysiology of human LCA8 caused by Crb1 mutations [26]. Host retinas were first examined at P21 or P35 in retinal flat-mount preparations using stacked images obtained with a confocal microscope. In general, the areas occupied by engrafted eGFP (+) cells varied greatly ( Fig. 1A; P21 (n > 3) and P35 (n > 5)), and transplanted cells displayed  (Fig. 1B). The most prevalent eGFP (+) cell type had a characteristic Hoechst 33258 staining pattern resembling that of rods [29,30] and a strong eGFP signal. These cells also showed two to three brightly stained chromocenters, strong cytoplasmic eGFP signal and bipolar processes with termini (Fig. 1B1). The other cell type was typically larger than rods and demonstrated a weak eGFP signal and several small chromocenters, which is a characteristic of INL cells (Fig. 1B1). Confocal analysis and 3D reconstruction of images from Pals1 CKO showed that transplanted eGFP (+) cells formed multi-layered cell clumps (from a single cell to 3-5 layered cells in thickness), and cellular processes in host retinas similar in structure to synaptic termini ( Fig. 1 B3 -B7). In parallel, to compare the survival of eGFP (+) transplanted cells in control animals, retinas of SW pups received grafts that were injected, harvested and examined in a similar fashion. As in Pals1 CKO, eGFP (+) cell clumps showed two nuclear Hoechst 33258 staining patterns and two eGFP staining patterns ( Fig. 1C -D2). In addition, eGFP (+) transplanted cells formed multi-layered clumps, and extensive bipolar processes and synaptic termini, which resembled the features of photoreceptor cells (Fig. 1D3 -D6). In summary, multi-layered grafted eGFP+ retinal cells similarly formed two distinctive patterns following transplantation into either SW or Pals1 CKO hosts.

Retinal integration
We next studied the profile of retinal migration of the donor cells in Pals1 CKO. In order to assess the progressive changes, paraffin sections of retinas were analyzed at P21, P35 and P60. Examination of donor cells in P21 Pals1 CKO retinas, when primary retinal development is completed, revealed two different phenotypes. In the first phenotype, a subset of eGFP (+) transplanted cells had migrated in the ONL ( Fig. 2A -A1), but most remained in the subretinal space. This phenotype occurred in areas of Pals1 CKO retina that appeared normal, where Muller glial arrangement was unaltered compared to WT, and which clearly differed from areas of Pals1 CKO retina that were severely affected ( Fig. 2A -C). For example, the location and distribution of Muller cell bodies (Sox9 (+)) in this unaffected Pals1 CKO retinal region resembled WT more closely than they resembled the severely affected Pals1 CKO region, where Muller glial cell bodies were apically displaced and the apical surface, including OLM, was severely disorganized [26]. Mosaic expression of Cre in Rx-Cre driver most likely accounted for the phenotypic variation in Pals1 CKO [26]. Migrated cells formed structures reminiscent of rod cells, including bipolar processes and rod termini, at the bottom of ONL.
In the other predominant phenotype, a majority of donor cells failed to migrate into the host retina (Fig. 2D). Instead, they formed clumps between retinal rosettes and RPE or in or around superficial retinal areas separated by Hoechst 33258 staining (blue) is shown for nuclear counterstaining. Scale bars, 50 μm rosettes ( Fig. 2D1-D2). The latter phenotype preferentially appeared around retinas undergoing active rearrangement exemplified by folding or rosette formation at P21. The predominantly subretinal retention of donor cells was largely unaltered at later stages (at P35 (n > 5) and P60 (n = 3)) ( Fig. 2E & F).

Differentiation of transplanted cells
We next examined the extent of retinal differentiation by immunofluorescent antibody staining of paraffin sections. Most eGFP (+) cells that migrated into Pasl1 CKO retinas were morphologically similar to rods: bipolar processes emanated from the cell body, and the location and shape of the termini were consistent ( Fig. 2A -A1). We also examined clumped cells that failed to migrate into the retina with retinal cell-type specific markers. Most of the donor cells expressed the rod-specific gene, rhodopsin (Fig. 3ac), and a subset of eGFP (+) rod cells showed the postsynaptic marker, PSD-95, in close proximity, suggesting maturation into rod photoreceptor cells (Fig. 3d). A subset of cells that remained in the subretinal space expressed glutamine synthetase (GS), a marker of Muller glia (Fig. 3eh). However, transplanted eGFP (+) Muller cells did not extend apical and basal processes encompassing the entire retina. Similarly, a small number of clumped cells expressed Chx10 (bipolar cells) (Fig. 3il) or Pax6 (ganglion, amacrine and horizontal cells; result not shown). eGFP (+)/Chx10 (+) or eGFP (+)/Pax6 (+) cells were not fully integrated into the recipient retina and were mainly localized in the subretinal space.
Host-mediated responses to cell transplantation eGFP (+) injected cells appeared to differentiate relatively normally within the subretinal space of the host (Fig. 3). This may be due to the intrinsic properties of the donor cells, which were enriched with late-stage progenitors and rod precursors, and to the inner-retinal environment of the neonatal host, which favors neurogenesis and neural differentiation. Some eGFP (+) injected cells integrated into the ONL of the recipient retina, but most remained in the subretinal space. Although a previous study showed that retinal integration largely occurs within 4 days posttransplantation in adult host mice [20], this may be caused by insufficient time for retinal migration after neonatal injection in neonatal LCA8-like model. When multiple transplanted clusters were examined at P35 (n > 5) and P60 (n = 3), retinal integration was not altered (Fig. 2). The non-permissive host environment provides another explanation. For example, host immune responses and the disease-specific host environment, alone or in combination, may significantly affect the fates of the injected cells. In order to assess these influences, two different retinal regions of Pals1 CKO, one normal looking and the other actively degenerating, were stained for evidence of MG activation and chondroitin sulfate proteoglycan (CSPG) induction at P21. To investigate MG activation, which plays a significant role in inhibiting effective cell transplantation [31], retinal sections were stained with α-Iba I antibody, which detects both resting and pathologically activated M1 MG [32][33][34]. In normal looking regions of the Pals1 CKO retinas, a small number of MG cells had been recruited by transplanted cells (Fig. 4a -c). In degenerating regions of Pals1 CKO retinas, Iba I (+) MG cells were intermixed with the injected cells ( Fig. 4eg, inset). The MG cells ectopically recruited in the subretinal space were hypertrophic, suggesting possible activation into the M1 stage. Therefore, it is likely that injected donor cells induced the recruitment, and possibly the activation, of MG cells in host retina. In parallel, normal SW retinas also attracted MG cells in and around grafted cells ( Fig. 4ik, insets).
We also examined the involvement of CSPG, a potent inhibitor of cell survival and migration that is normally expressed in the interphotoreceptor matrix [31,35,36]. When chondroitinase ABC was administered into the subretinal space of Rho -/mice simultaneously with a transplant, retinal migration was facilitated by breakdown of the glial scar and CSPGs [37]. Analysis of α-CS-56 immunoreactivity (specific to chondroitin sulfate types A and C, but not B) revealed that the CSPG level was lower in the host area filled with injected eGFP (+) cells than the level of endogenous CSPG in the interphotoreceptor matrix of the transplanted and nontransplanted neighboring areas (Fig. 4a, d, e and h). In order to assess whether this pattern of CSPG induction is a general feature of host retinas, we also examined SW retinas. SW retinas, when examined at P13 (n = 2) after neonatal transplantation of eGFP (+) cells, showed more robust CSPG induction than Pals1 CKO retinas, suggesting host-specific differences (Fig. 4il). In summary, the pathological environment in Pals1 CKO and SW retinas elicits MG recruitment upon cell transplantation, but Pals1 CKO retinas do not induce vigorous CSPG expression.

Cellular rearrangement in Pals1 CKO during and after retinal development
Although induction of the MG-mediated immune response may affect survival of the transplanted cells, other inhibitory mechanisms may also play significant roles in causing integration to be much less efficient in degenerating regions of Pals CKO retina than in WT or non-affected regions. One potential mechanism is the despite the apparent rosette forming activity at P0 (Fig. 5ad). At P22, many INL cells located among retinal rosettes were positioned apically compared to WT controls, and a few were located at the apical surface (Fig. 5e & f). Five-month old Pals1 CKO retinas showed GCL cells and, to a lesser degree, INL cells, abnormally localized at the apical surface (Fig. 5g & h). These observations are consistent with our previous report showing partial duplication of the retina in postnatal retinas of Pals1 CKO [26].
In summary, host retinal properties of Pals1 CKO may impose two major inhibitory barriers to transplanted cells. First, potentially pathological MG cells are recruited to the injected site. In addition, retinal cellular arrangement during rosette formation may oppose a strong inhibitory force to the retinal integration of transplanted cells. Because subretinal cell injection induces CSPG in SW, but not in Pals1 CKO, intrinsic properties of the host retina and responses to the transplanted cells

Discussion
LCA8 is unique among the approximately 20 subtypes of LCA in that it is caused by mutations in apical polarity complex gene, Crb1 [1,2,24,37]. As a result, affected retinas show destabilized OLM, pseudorosettes and thickening of the central retina (parafovea). Intriguingly, most of the human phenotype is recapped in mouse mutants not only of Crb1 gene, but also of Crb2, homolog and Pals1, interacting protein [24][25][26]. It is also interesting that human Crb1 mutations located at extracellular and intracellular domains induce milder late-onset RP12 or severe early-onset LCA8 without an obvious genotype-phenotype correlation [21]. Although the onset and severity of these two diseases are significantly different, both are caused by defects in retinal structural integrity. In rd 8 /rd 8 , a spontaneous frame-shift mutant of Crb1 and a mouse model for RP12, retinal lesions are focal and caused by failure to form cellto-cell attachment between rod photoreceptor cells and Muller glia [9,11]. In other mouse models partially mimicking human LCA8 pathology, abnormalities are observed in early embryonic retinas. Because the genesis of the majority of the rods and Muller glia starts postnatally [38,39], retinal laminar disorganization is likely caused by attachment failure between progenitor cells. Also, in contrast to RP12, in LCA8 the initial cellular detachment occurs in developing retina while cells are born and migrate via interkinetic nuclear migration, and while the retina is growing horizontally. The extensive horizontal growth of the retina can magnify the effects of loss of cellular attachments.
Examination of whole-mount sections in the present study shows that eGFP (+) retinal cells, which contain late-stage progenitors, precursors of rods and Muller glia and late-born amacrine cells in addition to postmitotic retinal neurons, form clumps whose area varies enormously in Pals1 CKO and SW retinas. The size of the clumps is presumably affected by subretinal targeting efficiency and survival of the transplanted cells. Therefore, we analyzed the fates of the transplanted cells and host responses qualitatively rather than quantitatively. We found that host retinal organization greatly influenced retinal integration of transplanted cells; unaffected or partially affected Pals1 CKO retinas showed facilitated migration of eGFP (+) cells, whereas migration was severely inhibited in retinal areas dominated by rosettes and/or laminar disorganization. Cells in the clumps expressed characteristic retinal markers, such as rhodopsin (rods), Pax6 (amacrine, horizontal and ganglion cells), Chx10 (bipolar cells) and GS (Muller glia). This expression pattern can be interpreted as evidence that transplanted cells clumped in the subretinal space differentiated normally. This view is supported by our observation of the terminal rod marker, PSD-95, in subsets of the transplanted cells and of rod cell processes and synaptic termini in 3D reconstruction images of the grafts. However, because Pax6 and Chx10 are expressed in retinal progenitors and precursors of retinal interneurons and bipolar cells, this result may simply suggest that donor cells within the graft maintain retinal gene expression. Despite the terminal differentiation, the morphology of the grafted clumped cells in general was severely defective. GS (+) Muller glial cells in the clumps, for example, did not demonstrate bipolar processes or expanded endfeet. Nevertheless, these injected cells were contained in the subretinal space of the host retinas throughout the stages examined, up to P60, more than 55 days after injection. Therefore, it is highly likely that the host-specific disease environment affects the migration of the injected cells. Factors suppressing the effectiveness of the cell transplantation therapy include the host immune response and activation of cell death, in addition to intrinsic properties of the donor cells.
One formidable host response is MG-activation, which not only inhibits donor cell integration into the retina, but also triggers apoptotic processes by a pro-inflammatory mechanism [40,41]. Diseased host retinas such as Pals1 CKO show the signs of MG activation as early neonates. Breakdown products of retinal cells at the apical surface may play a direct role in recruiting resting MGs. Heightened immune surveillance by the diseased host in response to cell transplantation may therefore supplement the inhibition due to donor cells, which can facilitate pathogenic activation of MG cells via cellular proteins, fragments and debris. In addition, inherent rosette forming activity in developing Pals1 CKO retinas plays an important role in suppressing retinal integration of the transplanted cells. Tracing of layer-specific cells in Pals1 CKO retinas during disease progression showed the preferred apical positioning of the resident cells in degenerating retinas, especially in the areas between rosettes. It is conceivable that ONL cells in Pals1 CKO, which are held together by OLM after random breakdown of cell attachments, form a rosette when the developing retina grows horizontally. To a lesser degree, INL and GCL cells, which are in the process of forming columnar circuits, also responded to rosette formation mediated by ONL by shifting their position apically. In extreme cases GCL cells were located at the apical, rather than the basal, side of the diseased retina. This interpretation is consistent with the observation of ectopic Pax6 and β-Tubulin III (+) cells in the apical surface of the retinas by immunofluorescence antibody staining [26].
During retinal development in LCA8, cell-to-cell adhesion is lost at the apical surface of the epithelium due to destabilization of Crumbs polarity complex [26]. Contact between retinal progenitor cells can be lost at an early stage, but contact between photoreceptor cells and Muller glia can also be lost at a late stage and after development is complete. In LCA8 and LCA8 animal models, the initial defects start during embryonic development (Fig. 6). When the loss of cellular attachment is extensive, ONL cells located between detachment points start to form a half-rosette due to horizontal retinal growth and apical constrictions among cells holding together. This process is further intensified to form a full rosette as development continues and results in delayed or abnormal photoreceptor maturation. The size of the rosettes varies and is determined by the distance between the two points at which extensive attachment is initially lost; there may be further division into smaller rosettes as more cellular detachment occurs within rosettes. During ONL rearrangement, INL cells and, to a lesser extent, GCL cells, are pushed apically through the gaps between rosettes [42], so that the retina not only loses laminar integrity but also fails to maintain proper circuitry. It is speculated that degeneration of retinal cells, including photoreceptors, is enhanced by abnormal oxidative stress. This stress is presumably imposed by excess oxygen due to decreased oxygen consumption and by improper nutrition due to secondary defects in blood supply. Retinal cellular rearrangement initiated by defects in cell-to-cell attachment may therefore impose an additional LCA8-specific inhibitory environment for cell transplantation therapy. Our results contrast dramatically with earlier reports demonstrating facilitated cell migration after retinal injection in animal models with partial breakdown of OLM [8]. These studies were performed in adult mice, in which mature retina may not exhibit significant rearrangement in response to localized cell-to-cell junctional breakdown, rather than in neonatal LCA8 retinas, in which robust developmental rearrangement follows retinal growth. The results with Pals1 CKO LCA8-like hosts also differed from those with SW hosts. In areas of SW retinas where rosettes were not formed, grafts both recruited MG and induced CSPG. However, in Pals1 CKO retinas, grafts attracted Iba1 (+) MG, but CSPG induction was minimal. This difference supports the notion that, in addition to undergoing the intrinsic cellular rearrangements that form retinal rosettes, the Pals1 CKO retina provides an environment with a distinctive host response to transplanted cells.

Conclusions
This study identifies disease-specific factors that affect the survival and retinal integration of transplanted cells in the early-onset degenerative retinal disease LCA8. In addition to the general inhibitory host responses, such as MG activation, the neonatal LCA8 environment may impose a physical restraint due to cellular rearrangement in a degenerating retina with partly broken junctions essential for tissue integrity. Customized interventions designed to overcome these inhibitory host barriers will be essential for successful ocular cell-based therapy for LCA8.


extfieldsofstudy: Biology Medicine

text:
Siegesbeckia orientalis Extract Inhibits TGFβ1-Induced Migration and Invasion of Endometrial Cancer Cells

Type II endometrial carcinoma typically exhibits aggressive metastasis and results in a poor prognosis. Siegesbeckia orientalis Linne is a traditional Chinese medicinal herb with several medicinal benefits, including the cytotoxicity against various cancers. This study investigates the inhibitory effects of S. orientalis ethanol extract (SOE) on the migration and invasion of endometrial cancer cells, which were stimulated by transforming growth factor β (TGFβ). The inhibitory effects were evaluated by determining wound healing and performing the Boyden chamber assay. This study reveals that SOE can inhibit TGFβ1-induced cell wound healing, cell migration, and cell invasion in a dose-dependent manner in RL95-2 and HEC-1A endometrial cancer cells. SOE also reversed the TGFβ1-induced epithelial-mesenchymal transition, including the loss of the cell-cell junction and the lamellipodia-like structures. Western blot analysis revealed that SOE inhibited the phosphorylation of ERK1/2, JNK1/2, and Akt, as well as the expression of MMP-9, MMP-2, and u-PA in RL95-2 cells dose-dependently. The results of this investigation suggest that SOE is a potential anti-metastatic agent against human endometrial tumors.

Introduction
Endometrial cancer is a common gynecologic malignancy in the Western world [1,2]. In Taiwan, the incidence of endometrial cancer is second only to that of cervical cancer in women and the number of cases is rising gradually [3]. Endometrial cancer can be classified into Type I and Type II based on clinical behavior and morphological phenotype. Type I cases are low-stage and low-grade, while Type II cases are advanced-stage and aggressive [4]. Most cases of Type I can be diagnosed at an early stage because some symptoms would appear in this stage, and such cases present a high survival percentage following primary surgery. In contrast, Type II patients typically have a poor prognosis because the carcinoma has an aggressive phenotype that is characterized by lymphovascular invasion, high histological grade, and myometrial invasion, resulting in distant metastases via the hematogenous route [5].
Transforming growth factor β (TGFβ) signaling has been identified as crucial in the initial steps of endometrial carcinoma invasion and metastasis [6]. The function of TGFβ in tumor biology is complex. It suppresses tumor activity in the early stages of carcinogenesis, but becomes a tumor promoter in the later stages [7]. Three TGFβ isoforms (β1, β2, and β3) exists in human endometrial tumors. TGFβ1 can activate tumor promotion and the epithelial-to-mesenchymal transition (EMT), which makes tumor cells move away from their epithelial cell community and integrate into surrounding tissue [8][9][10]. Clinical studies have shown that a significant increase in levels of TGFβ1 in the serum of patients with breast cancer, lung cancer, hepatoma, prostate cancer, and stage I and stage II endometrial carcinoma [11,12]. The overexpression of TGFβ1 in endometrial cancer cells correlates with tumor metastasis and a poor patient outcome [13,14].
For patients in an advanced or later stage endometrial cancer, despite surgery, treatment includes chemotherapy, radiotherapy, and hormonal treatment [15]. Chemotherapy with cisplatin, doxorubicin, and paclitaxel has been found to be superior to radiotherapy [16]. However, the effectiveness of these strategies remains limited. Recently, several reports have suggested that Chinese herbal medicines are effective in delaying tumor progression, preventing recurrence and metastasis, alleviating clinical symptoms, improving immune function, increasing the quality of life, and prolonging the life span of cancer patients [17,18].
Siegesbeckia orientalis L. is a member of the Asteraceae family. As a potential and popular folk-medicine herb, S. orientalis has been prescribed for treating snakebites, cutaneous disorders, rheumatic arthritis [19,20], allergies [21], and immune deficiency [22], and it is also taken orally as an anti-inflammation and anti-cancer agent [23,24]. The authors' laboratory has confirmed, using in vitro and in vivo models, that the ethanol extract of S. orientalis (SOE) can attenuate acute inflammation by inhibiting inflammatory mediators through the suppression of MAPKs-and NF-κB-dependent pathways [25]. We have also demonstrated that SOE inhibits the growth of RL95-2 human endometrial cancer cells in a dose-dependent manner, and this effect is associated with G2/M phase cell cycle arrest, activation of caspase-3, -8, and -9, upregulation of Bad, Bak, and Bax, and downregulation of Bcl-2 and Bcl-xL [26]. The present study investigates the inhibitory effects of SOE on the motility and invasion of endometrial cancer cells under the stimulation by TGFβ1. The effects of SOE on the expression of MMPs, and the activities of MAPK and Akt were also examined.

Effect of SOE on Morphology of Endometrial Cancer Cells
TGFβ1 may result in the acquisition of an invasive phenotype of endometrial carcinoma. The mesenchymal phenotype is an important characteristic of EMT and is associated with invasive and metastatic effects [6]. As shown in Figure 1, when 2.5 ng/mL of TGFβ1 was added to endometrial cancer cells, the mesenchymal phenotype was changed, including the loss of the cell-cell junction and the formation of a lamellipodia-like structure. Under the induction by TGFβ1, the treatment of SOE reversed the mesenchymal phenotypes of HEC-1A and RL95-2 cancer cells.

Inhibitory Effect of SOE on Proliferation of Endometrial Cancer Cells
The inhibitory effects of treatment with various SOE concentrations for various incubation durations on the viability of HEC-1A and RL95-2 cells were examined. Figure 2 reveals that SOE, with or without TGFβ1 induction, inhibited the viability of both cell lines in dose-and time-dependent manner. Treatment for 24-48 h with SOE concentrations of 0 to 25 µg/mL did not significantly alter the growth of these two cell lines relative to the vehicle (treated with DMSO alone). To eliminate the influence of cell death on observed parameters of cell migration and invasion, this concentration range was used in all subsequent experiments. Cancer cells were cultured in 96-well plates at a density of 1 × 10 4 cells/well. Cells were treated with 0-150 μg/mL SOE for 24-72 h. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. Three independent experiments were conducted; results are expressed as mean ± SD. A significant difference from the respective vehicle, according to Student's t-test, was indicated as "a" for p < 0.05; "b" for p < 0.01, and "c" for p < 0.001.

Inhibitory Effect of SOE on Migration of Endometrial Cancer Cells
The inhibitory effects of SOE on the motility of HEC-1A and RL95-2 cells were firstly studied using the wound healing assay. When treated with TGFβ1 in the absence of SOE (0 μg/mL), cells of both lines moved rapidly into the gap (Figure 3). Exposing the cells to a high concentration of SOE (15 and 20 μg/mL), with or without TGFβ1 induction, reduces their ability to migrate. These results indicate that SOE inhibited the migration of both cell lines in a dose-dependent manner, even under the induction with TGFβ1.
A cell migration assay that used the Boyden chamber with a polycarbonate membrane was utilized to study further the effect of SOE on the motility of HEC-1A and RL95-2 cells. Cells were plated in the upper chamber, and the cells that moved to the underside of the coated polycarbonate membrane were counted under an optical microscope. SOE treatment significantly reduced the number of cells that migrated to the lower chamber ( Figure 4). SOE retarded the migration of HEC-1A and RL95-2 cells in a dose-dependent manner relative to that of untreated controls. Cancer cells were cultured in 96-well plates at a density of 1ˆ10 4 cells/well. Cells were treated with 0-150 µg/mL SOE for 24-72 h. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Three independent experiments were conducted; results are expressed as mean˘SD. A significant difference from the respective vehicle, according to Student's t-test, was indicated as "a" for p < 0.05; "b" for p < 0.01, and "c" for p < 0.001.

Inhibitory Effect of SOE on Migration of Endometrial Cancer Cells
The inhibitory effects of SOE on the motility of HEC-1A and RL95-2 cells were firstly studied using the wound healing assay. When treated with TGFβ1 in the absence of SOE (0 µg/mL), cells of both lines moved rapidly into the gap ( Figure 3). Exposing the cells to a high concentration of SOE (15 and 20 µg/mL), with or without TGFβ1 induction, reduces their ability to migrate. These results indicate that SOE inhibited the migration of both cell lines in a dose-dependent manner, even under the induction with TGFβ1.
A cell migration assay that used the Boyden chamber with a polycarbonate membrane was utilized to study further the effect of SOE on the motility of HEC-1A and RL95-2 cells. Cells were plated in the upper chamber, and the cells that moved to the underside of the coated polycarbonate membrane were counted under an optical microscope. SOE treatment significantly reduced the number of cells that migrated to the lower chamber ( Figure 4). SOE retarded the migration of HEC-1A and RL95-2 cells in a dose-dependent manner relative to that of untreated controls. For the cells induced by TGFβ1, motility decreased to 25.2% (HEC-1A cells) and 30.1% (RL95-2 cells) after treatment with 20 µg/mL SOE for 48 h (p < 0.001). Cancer cells were cultured in 96-well plates at a density of 1 × 10 4 cells/well. Cells were treated with 0-150 μg/mL SOE for 24-72 h. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. Three independent experiments were conducted; results are expressed as mean ± SD. A significant difference from the respective vehicle, according to Student's t-test, was indicated as "a" for p < 0.05; "b" for p < 0.01, and "c" for p < 0.001.

Inhibitory Effect of SOE on Migration of Endometrial Cancer Cells
The inhibitory effects of SOE on the motility of HEC-1A and RL95-2 cells were firstly studied using the wound healing assay. When treated with TGFβ1 in the absence of SOE (0 μg/mL), cells of both lines moved rapidly into the gap (Figure 3). Exposing the cells to a high concentration of SOE (15 and 20 μg/mL), with or without TGFβ1 induction, reduces their ability to migrate. These results indicate that SOE inhibited the migration of both cell lines in a dose-dependent manner, even under the induction with TGFβ1.
A cell migration assay that used the Boyden chamber with a polycarbonate membrane was utilized to study further the effect of SOE on the motility of HEC-1A and RL95-2 cells. Cells were plated in the upper chamber, and the cells that moved to the underside of the coated polycarbonate membrane were counted under an optical microscope. SOE treatment significantly reduced the number of cells that migrated to the lower chamber ( Figure 4). SOE retarded the migration of HEC-1A and RL95-2 cells in a dose-dependent manner relative to that of untreated controls. For the cells induced by TGFβ1, motility decreased to 25.2% (HEC-1A cells) and 30.1% (RL95-2 cells) after treatment with 20 μg/mL SOE for 48 h (p < 0.001).
(A)  After the culture insert was removed, DMEM with specified SOE concentrations was added, with or without TGFβ1 (2.5 ng/mL) stimulation, and the cells were then incubated. Photomicrographs of culture plates were directly obtained using a phase-contrast microscope (magnification 20×). Closure rate is defined as 100% without TGFβ1 induction and without SOE treatment. Three independent experiments were conducted; results are expressed as mean ± SD. A significant difference from the respective vehicle, according to Student's t-test, was indicated as "a" for p < 0.05; "b" for p < 0.01, and "c" for p < 0.001. After the culture insert was removed, DMEM with specified SOE concentrations was added, with or without TGFβ1 (2.5 ng/mL) stimulation, and the cells were then incubated. Photomicrographs of culture plates were directly obtained using a phase-contrast microscope (magnification 20ˆ). Closure rate is defined as 100% without TGFβ1 induction and without SOE treatment. Three independent experiments were conducted; results are expressed as mean˘SD. A significant difference from the respective vehicle, according to Student's t-test, was indicated as "a" for p < 0.05; "b" for p < 0.01, and "c" for p < 0.001. After the culture insert was removed, DMEM with specified SOE concentrations was added, with or without TGFβ1 (2.5 ng/mL) stimulation, and the cells were then incubated. Photomicrographs of culture plates were directly obtained using a phase-contrast microscope (magnification 20×). Closure rate is defined as 100% without TGFβ1 induction and without SOE treatment.
Three independent experiments were conducted; results are expressed as mean ± SD. A significant difference from the respective vehicle, according to Student's t-test, was indicated as "a" for p < 0.05; "b" for p < 0.01, and "c" for p < 0.001.
(A)  Cells that penetrated the polycarbonate membrane to lower surface of the filter were stained with 0.5% crystal violet. The cell numbers were visualized under an inverted microscope with a magnification of 100× and were counted per field. The migration index is defined as 100% without TGFβ1 induction and without SOE treatment. Three independent experiments were performed; results are expressed as mean ± SD. A significant difference from the respective vehicle, according to Student's t-test, was indicated as "a" for p < 0.05, "b" for p < 0.01, and "c" for p < 0.001.

Inhibitory Effect of SOE on Invasion of Endometrial Cancer Cells
The progress of tumor invasion includes the degradation of basement membranes, the proteolysis of extracellular matrix (ECM), pseudopodial extension, and cell migration. The basement membrane, which is the first barrier of ECM to invasion by cancer, is composed of matrix macromolecules, such as type IV collagen, heparan sulfate proteoglycans, and laminin [27]. Thus, a Boyden chamber with a polycarbonate membrane that is coated with a collagen layer was used to investigate the inhibitory effect of SOE on invasion of endometrial cancer cells. SOE treatment of both cell lines, with and without TGFβ1 induction, significantly reduced the number of cells from the upper chamber that invaded the lower chamber ( Figure 5). Quantification of cells in the lower chamber demonstrates that SOE treatment significantly inhibited the invasion of both cell lines in a dose-dependent manner. For the cells induced by TGFβ1, cell invasion was reduced to 14.4% (HEC-1A cells) and 1.7% (RL95-2 cells) after treatment with 20 μg/mL SOE for 48 h (p < 0.001). The migration assay was conducted using the Boyden chamber. HEC-1A and RL95-2 cells were treated with various doses of SOE for 48 h. Cells that penetrated the polycarbonate membrane to lower surface of the filter were stained with 0.5% crystal violet. The cell numbers were visualized under an inverted microscope with a magnification of 100ˆand were counted per field. The migration index is defined as 100% without TGFβ1 induction and without SOE treatment. Three independent experiments were performed; results are expressed as mean˘SD. A significant difference from the respective vehicle, according to Student's t-test, was indicated as "a" for p < 0.05, "b" for p < 0.01, and "c" for p < 0.001.

Inhibitory Effect of SOE on Invasion of Endometrial Cancer Cells
The progress of tumor invasion includes the degradation of basement membranes, the proteolysis of extracellular matrix (ECM), pseudopodial extension, and cell migration. The basement membrane, which is the first barrier of ECM to invasion by cancer, is composed of matrix macromolecules, such as type IV collagen, heparan sulfate proteoglycans, and laminin [27]. Thus, a Boyden chamber with a polycarbonate membrane that is coated with a collagen layer was used to investigate the inhibitory effect of SOE on invasion of endometrial cancer cells. SOE treatment of both cell lines, with and without TGFβ1 induction, significantly reduced the number of cells from the upper chamber that invaded the lower chamber ( Figure 5). Quantification of cells in the lower chamber demonstrates that SOE treatment significantly inhibited the invasion of both cell lines in a dose-dependent manner. For the cells induced by TGFβ1, cell invasion was reduced to 14.4% (HEC-1A cells) and 1.7% (RL95-2 cells) after treatment with 20 µg/mL SOE for 48 h (p < 0.001). The cell numbers were visualized under an inverted microscope with a magnification of 100× and were counted per field. The invasion index is defined as 100% without TGFβ1 induction and without SOE treatment. Three independent experiments were performed; results are expressed as mean ± SD. A significant difference from the respective vehicle, according to Student's t-test, was indicated as "a" for p < 0.05; "b" for p < 0.01; and "c" for p < 0.001.

Effects of SOE on Related Proteins Expression in RL95-2 Cells
The progress of tumor metastasis is regulated by complex mechanisms. The degradation of the ECM by increasing the expression and activation of extracellular proteases, such as metalloproteinases (MMPs), urokinase-type plasminogen activator (u-PA), or serine proteinase, has been regarded as important in cancer invasion and metastatic processes [28]. MMPs are the most essential proteases in the proteolysis of ECM proteins, such as collagen, fibronectin, proteoglycan, laminin, and elastin [29]. MMPs also play important roles in differentiation, proliferation, angiogenesis, and the destruction of The cell numbers were visualized under an inverted microscope with a magnification of 100ˆand were counted per field. The invasion index is defined as 100% without TGFβ1 induction and without SOE treatment. Three independent experiments were performed; results are expressed as mean˘SD. A significant difference from the respective vehicle, according to Student's t-test, was indicated as "a" for p < 0.05; "b" for p < 0.01; and "c" for p < 0.001.

Effects of SOE on Related Proteins Expression in RL95-2 Cells
The progress of tumor metastasis is regulated by complex mechanisms. The degradation of the ECM by increasing the expression and activation of extracellular proteases, such as metalloproteinases (MMPs), urokinase-type plasminogen activator (u-PA), or serine proteinase, has been regarded as important in cancer invasion and metastatic processes [28]. MMPs are the most essential proteases in the proteolysis of ECM proteins, such as collagen, fibronectin, proteoglycan, laminin, and elastin [29]. MMPs also play important roles in differentiation, proliferation, angiogenesis, and the destruction of inflammatory tissue [30,31]. Among the MMPs, MMP-2 (gelatinase A), and MMP-9 (gelatinase B) are the most essential in degrading of type IV collagen, which is the main constituent of the basement membrane [30]. Numerous papers have reported that the expressions of MMPs and u-PA correlate closely with the migration, invasion, and angiogenesis of various tumors [28][29][30]32].
Mitogen-activated protein kinases (MAPKs) are known to mediate and regulate cell growth, differentiation, apoptosis, and metastasis. The MAPK members, ERK1/2, p38 MAPK, and JNK/SAPK, have a critical role in regulating the expression of MMPs and u-PA [28,29,31,32]. Furthermore, the PI3K/Akt signaling pathway also plays a vital role in the regulation of MMPs and u-PA expression, and this pathway is associated with cell survival and metastasis of the development and progression of various tumors [29,[32][33][34][35]. Recent studies have demonstrated that MAPK and PI3K/Akt signaling pathways are involved in the migration and metastasis of endometrial cancer cells [36,37]. inflammatory tissue [30,31]. Among the MMPs, MMP-2 (gelatinase A), and MMP-9 (gelatinase B) are the most essential in degrading of type IV collagen, which is the main constituent of the basement membrane [30]. Numerous papers have reported that the expressions of MMPs and u-PA correlate closely with the migration, invasion, and angiogenesis of various tumors [28][29][30]32]. Mitogen-activated protein kinases (MAPKs) are known to mediate and regulate cell growth, differentiation, apoptosis, and metastasis. The MAPK members, ERK1/2, p38 MAPK, and JNK/SAPK, have a critical role in regulating the expression of MMPs and u-PA [28,29,31,32]. Furthermore, the PI3K/Akt signaling pathway also plays a vital role in the regulation of MMPs and u-PA expression, and this pathway is associated with cell survival and metastasis of the development and progression of various tumors [29,[32][33][34][35]. Recent studies have demonstrated that MAPK and PI3K/Akt signaling pathways are involved in the migration and metastasis of endometrial cancer cells [36,37].  The expressed protein levels of p-ERK, p-JNK, p-p38, p-Akt, MMP-9, MMP-2, and u-PA following SOE treatment were evaluated by Western blotting. Regardless of whether the RL95-2 cells were stimulated by TGFβ1, Western blot analysis demonstrates that SOE treatment significantly reduced the levels of proteins p-ERK (42/44 kDa), p-JNK (46/54 kDa), p-Akt (60 kDa), MMP-9 (92 kDa), MMP-2 (64/72 kDa), and u-PA (33 kDa) in a dose-dependent manner ( Figure 6). The SOE concentration range (5 to 20 µg/mL) that inhibited the expression of MAPKs, p-Akt, and MMPs coincided with the kinetics of cell migration and invasion.

Materials
The S. orientalis L. plant materials were purchased from Yuanshan Company (Kaohsiung City, Taiwan). Its nucleotide sequence was determined and deposited in the GenBank database with accession number JN987228 [38].

Preparation of S. orientalis Ethanol Extract
The preparation of SOE was conducted as described previously [25]. In brief, dried powder (9.3 kg) of the aerial part of S. orientalis L. was extracted with 47 L of 95% ethanol for one day and this process was repeated three times. The extracted solutions were collected and filtered, and the solvent was removed using a rotary evaporator. The residue was then dried in a freeze-dryer. Total dry mass of this extract was 489 g. The chemical composition of the SOE has been reported elsewhere [26].

Cancer Cell Lines and Culture
Human endometrial cancer cell lines RL95-2 and HEC-1A were purchased from the American Type Cell Collection (ATCC, Manassas, VA, USA). Cells of these two cell lines were grown in DMEM that was supplemented with 10% (v/v) FBS, 1% penicillin/streptomycin and 0.02% sodium bicarbonate. These cancer cells were cultivated at 37˝C with 5% CO 2 and 95% air, and in 100% relative humidity. The SOE was dissolved in dimethyl sulfoxide (DMSO) and the final DMSO concentration in the medium was less than 0.1%.

Determination of Cytotoxicity for Cancer Cells
Cancer cells were cultured in 96-well plates at 2ˆ10 4 cells/well and treated with the indicated concentration of SOE. After cultivation for the given period, the medium solution was removed. A 100 µL volume of the culture medium that contained 0.5 mg/mL of the MTT assay kit was added to each well. The cells were further cultured for 2 h, and the medium solution was removed. An aliquot of 100 µL DMSO was added and the plate was shaken until the crystals dissolved. The cytotoxicity against cancer cells was measured at 570 nm using an ELISA reader (Model 550, Bio-Rad Laboratories, Hercules, CA, USA).

Cell Migration by Wound Healing Assay
Cancer cells were seeded at 1ˆ10 6 cells/mL in 200 µL DMEM with a Culture Insert-2 Well (ibidi GmbH, Munich, Germany) and were cultivated for 24 h. After the culture insert was removed, DMEM with specified SOE concentration was added, with or without TGFβ1 (2.5 ng/mL) stimulation, and the cells were then incubated. Images of unfilled areas under each condition were obtained using an inverted phase-contrast microscope (Nikon Eclipse TS100, Chiyoda-ku, Tokyo, Japan) at a magnification of 20ˆ. All wound healing assays were conducted in triplicate. The closure rate was calculated using the following formula: Closure rate p%q " pOriginal width´Width after migrationq{Original widthˆ100% (1)

Cell Migration and Invasion Assays by Boyden Chamber
Cell migration and invasion were assessed using the CytoSelect™ 24-well cell migration assay (Cell Biolabs, San Diego, CA, USA) and BioCoat™ Matrigel™ Invasion Chamber (BD Biosciences), respectively. To determine the effect of SOE on cell migration, 1ˆ10 6 cells/mL were seeded in serum-free DMEM on a polycarbonate membrane insert (8 µm pore size). The cells were then treated with various concentrations of SOE, with or without the stimulation by TGFβ1 (2.5 ng/mL). The bottom chamber was filled with DMEM that contained 10% FBS as a chemo-attractant. The chamber was incubated for 48 h at 37˝C. Next, non-migrating cells in the upper chamber were wiped away using a cotton-tipped swab, and invading cells were fixed with methanol, stained with 0.2% crystal violet, and photographed under a phase-contrast microscope. The same procedure was also carried out in experiments on cell invasion, except that the polycarbonate membrane was coated with a uniform layer of collagen. The migration index and the invasion index were estimated using the following equation: Migration index or invasion index " Nu{Ncˆ100% (2) Nu: number of cells that moved to the underside of the membrane following SOE treatment; Nc: number of cells that moved to the underside of the membrane without TGFβ1 induction and SOE treatment.

Western Blot Analysis
To analyze the related proteins, 1ˆ10 6 cells were seeded into 6-cm culture dishes with or without SOE treatment, and with or without TGFβ1 stimulation. Following 24 h of incubation, the medium was removed and cells were washed several times using phosphate-buffered saline (PBS, 0.01 M, pH 7.2). Whole-cell lysates were prepared as described previously [26]. The harvested protein concentration was measured using a protein assay kit (Bio-Rad). Samples with equal amounts of denatured proteins were resolved on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred onto a nitrocellulose membrane (Immunobilin P; Millipore, Billerica, MA, USA), and were blocked for 1 h using 10% skimmed milk. The membranes were incubated overnight at 4˝C with the indicated primary antibodies. On the next day, the primary antibodies were washed away and secondary antibodies were added for incubation for 1 h at room temperature. The protein levels were detected using Enhanced Chemiluminescence (ECL) Plus Western blotting detection reagents (Amersham Bioscience, Uppsala, Sweden). Densitometric analyses were conducted using the Quantity One ® software (Bio-Rad).

Statistical Analysis
All experiments were carried out for three to five independent replicates. The experimental data were analyzed by using Microsoft Excel software (Microsoft Software Inc., Redmond, WA, USA). The data are expressed in terms of mean and standard deviation, and the statistical differences were analyzed by Student's t-test.

Conclusions
In this study, SOE treatment effectively inhibited the migration and invasion of RL95-2 and HEC-1A endometrial cancer cells in a dose-dependent manner, with or without stimulation by TGFβ1, as determined by wound healing analysis and the Boyden chamber assay. SOE also reversed the TGFβ1-induced epithelial-mesenchymal transition. Western blot analysis demonstrated that SOE inhibited the phosphorylation of ERK1/2, JNK1/2, and Akt, as well as the expression of MMP-9, MMP-2, and u-PA in RL95-2 cells, in a dose-dependent manner. Since SOE also exhibits strong anti-inflammation and anti-proliferation activities, it may be a favorable complementary agent for treating patients with endometrial cancer.