s2fieldsofstudy: Biology Chemistry Medicine

text:
Silibinin suppresses EMT-driven erlotinib resistance by reversing the high miR-21/low miR-200c signature in vivo

The flavolignan silibinin was studied for its ability to restore drug sensitivity to EGFR-mutant NSCLC xenografts with epithelial-to-mesenchymal transition (EMT)-driven resistance to erlotinib. As a single agent, silibinin significantly decreased the tumor volumes of erlotinib-refractory NSCLC xenografts by approximately 50%. Furthermore, the complete abrogation of tumor growth was observed with the co-treatment of erlotinib and silibinin. Silibinin fully reversed the EMT-related high miR-21/low miR-200c microRNA signature and repressed the mesenchymal markers SNAIL, ZEB, and N-cadherin observed in erlotinib-refractory tumors. Silibinin was sufficient to fully activate a reciprocal mesenchymal-to-epithelial transition (MET) in erlotinib-refractory cells and prevent the highly migratogenic phenotype of erlotinib-resistant NSCLC cells. Given that the various mechanisms of resistance to erlotinib result from EMT, regardless of the EGFR mutation status, a water-soluble, silibinin-rich milk thistle extract might be a suitable candidate therapy for upcoming clinical trials aimed at preventing or reversing NSCLC progression following erlotinib treatment.

The flavolignan silibinin was studied for its ability to restore drug sensitivity to EGFR-mutant NSCLC xenografts with epithelial-to-mesenchymal transition (EMT)-driven resistance to erlotinib. As a single agent, silibinin significantly decreased the tumor volumes of erlotinib-refractory NSCLC xenografts by approximately 50%. Furthermore, the complete abrogation of tumor growth was observed with the co-treatment of erlotinib and silibinin. Silibinin fully reversed the EMT-related high miR-21/low miR-200c microRNA signature and repressed the mesenchymal markers SNAIL, ZEB, and N-cadherin observed in erlotinib-refractory tumors. Silibinin was sufficient to fully activate a reciprocal mesenchymal-to-epithelial transition (MET) in erlotinib-refractory cells and prevent the highly migratogenic phenotype of erlotinib-resistant NSCLC cells. Given that the various mechanisms of resistance to erlotinib result from EMT, regardless of the EGFR mutation status, a water-soluble, silibinin-rich milk thistle extract might be a suitable candidate therapy for upcoming clinical trials aimed at preventing or reversing NSCLC progression following erlotinib treatment.
T he discovery of somatic activating mutations in the epidermal growth factor receptor (EGFR) kinase domain has radically altered the treatment options of patients with non-small cell lung cancer (NSCLC) 1-5 . For example, Caucasian patients harboring activating mutations in the EGFR kinase domain, such as a deletion in exon 19 or L858R, have shown significant responses to the EGFR tyrosine kinase inhibitor (TKI) erlotinib, including a 70% response rate, 14-month progression-free survival, and 27-month median survival. Accordingly, the EGFR TKIs erlotinib or gefitinib have become the standard of care for first-line treatment in patients with advanced NSCLC and whose tumors harbor activating mutations in the kinase domain of EGFR. However, despite the encouraging clinical findings showing a favorable response to first-and second-line EGFR TKIs, advanced NSCLC patients with EGFR mutations often respond unpredictably to initial EGFR TKI therapy. Additionally, patients who initially show dramatic responses invariably relapse due to the development of drug resistance. Intriguingly, some NSCLC patients with wild-type EGFR also benefit from treatments with EGFR TKIs. Not surprisingly, numerous research groups are attempting to identify more effective predictive biomarkers for assessing the response or resistance to EGFR TKIs to optimize therapy for individual patients.
The acquisition of resistance to EGFR TKIs has been attributed to two main mechanisms reviewed in [6][7][8][9][10][11][12] . The first is the emergence of malignant clones containing second-site mutations in the EGFR kinase domain that abrogate the inhibitory activity of EGFR TKIs. The most common lesion is the so-called ''gatekeeper mutation'', which involves a substitution of methionine for threonine at position 790 (T790M). The second well-known mechanism of resistance to gefitinib or erlotinib is the amplification of the MET receptor tyrosine kinase (RTK) gene, which activates downstream intracellular signaling independently of EGFR. EGFR T790M and MET amplification account for approximately 60-70% of all known causes of acquired resistance to gefitinib or erlotinib; these amplifications are not mutually exclusive and can be detected in the same resistant tumor or may occur independently in different metastatic sites in the same patient. Consequently, ongoing research has been focused on identifying the molecular mechanisms accounting for the 30-40% of EGFR TKI-resistant, EGFR-mutant tumors that do not carry EGFR mutations or MET amplification 13 . In this regard, emerging evidence strongly suggests that the mesenchymal phenotype is closely related to the lack of responsiveness to EGFR TKIs [14][15][16][17][18][19][20][21][22][23][24][25] . First, although NSCLC cell lines with EGFR-activating mutations are the most sensitive to erlotinib, mesenchymal cell lines are highly resistant to erlotinib when compared to epithelial cell lines. Second, the correlation between an epithelial-to-mesenchymal transition (EMT) signature and greater resistance to erlotinib can be observed, even in EGFR wild-type gene cells exhibiting a mesenchymal-like phenotype. Third, genetic and histological analyses of tumor biopsies from NSCLC patients with acquired resistance to EGFR inhibitors have revealed that a subgroup of resistant carcinomas exhibit a pronounced EMT. Fourth, the activation of the receptor tyrosine kinase AXL, which confers acquired resistance to erlotinib in pre-clinical models of EGFR-mutated NSCLCs and is overexpressed in approximately 20% of post-erlotinib-treated patients with EGFR TKI resistance, has been associated with the development of an EMT phenotype. Fifth, in NSCLC, the EMT phenomenon does not appear to be a general marker of resistance toward commonly used chemotherapeutics, such as pemetrexed, docetaxel, and paclitaxel, as mesenchymal cells appear to be relatively more sensitive to cisplatin, gemcitabine, and vinorelbine. Sixth, an EMT signature that highlights the different patterns of drug responsiveness in epithelial and mesenchymal NSCLC cells has prognostic value for early-stage NSCLC and can also predict resistance to EGFR TKIs, regardless of the EGFR mutation status.
Emerging data have repeatedly implicated EMT in contributing to both innate and acquired resistance to erlotinib; accordingly, it might be tempting to suggest that EMT is a core resistance mechanism that might be targeted to bypass the inhibition of the EGFR pathway in NSCLC. Indeed, elucidating how EMT could be prevented or reversed in NSCLC should enable the development of new strategies aimed at optimizing the efficacy of EGFR TKIs to improve the therapeutic outcome of NSCLC patients. Although the inhibition of EMT can be a critical strategy for delaying or preventing tumor progression after treatment with EGFR TKIs, the reversal of the EMT-driven erlotinib-refractory phenotype is urgently needed to expand the current EMT-targeted therapies. In this regard, a landmark study showed that the combination of EGFR TKIs with the lignin silibinin, the bioactive constituent of silymarin isolated from the dried fruits of the milk thistle (Silybum marianum) plant [26][27][28][29] , drastically suppressed tumor growth. This was the case for both primary tumors and the cells that had acquired TKI resistance via the EGFR T790M mutation 30 . This finding might open a new, unexpected therapeutic avenue for the clinical management of EMT-driven, acquired resistance to erlotinib. Silibinin, a potent natural agent, appears to reverse EMT by decreasing the levels of key EMT transcription factors, such as SLUG, and increasing the expression levels of E-cadherin [31][32][33] . Because the ability of silibinin to overcome the erlotinib resistance that is attributed to mechanisms other than second-site EGFR mutations has not been explored and given that the poor water solubility and low bioavailability of silibinin might severely limit its clinical efficacy in NSCLC patients, we decided to explore whether a milk thistle extract rich in silibinin-meglumine, a commercially used water-soluble form of silibinin complexed with the excipient amino-sugar meglumine (Fig. 1), can reverse acquired resistance to EGFR TKIs in animal models. Here, we present the first evidence that, in the absence of second-site EGFR mutations or the activation of MET or AXL, a water-soluble formulation of the flavolignan silibinin, efficiently circumvents EMT-driven resistance to erlotinib via a mechanism that involves, at least in part, the restoration of the imbalance of such EMT-related microRNAs as miR-21 and miR-200c in erlotinib-refractory tumors in vivo.

Results
In the absence of second-site EGFR mutations, erlotinib-refractory, EGFR-mutant NSCLC mouse xenografts exhibit faster tumor growth than erlotinib-responsive xenografts. We subcutaneously implanted parental PC-9 cells and PC-9/Erl-R derivatives 34 in athymic nude mice to compare whether the acquisition of resistance to erlotinib significantly altered the tumor-forming ability of EGFR-mutant NSCLC cells. In the absence of erlotinib treatment, the PC-9/Erl-R cells exhibited significantly higher tumorigenicity than the parental PC-9 cells. The PC-9/Erl-R cells established larger tumors with shorter latencies in comparison to the parental PC-9 cells ( Supplementary Fig. 1). To confirm the genetic changes, which were previously described in cultured parental and resistant cells in vitro 34 , following the acquired resistance to erlotinib in vivo, we examined genomic DNA directly isolated from frozen tumors. We assessed the status of 85 possible mutations in the EGFR pathway using the Human EGFR Pathway qBiomarker Somatic Mutation PCR array, which combines allele-specific amplification and 59hydrolysis probe detection to detect as little as 0.01% somatic mutations in a background of wild-type genomic DNA. Similarly, we assessed the erlotinib-sensitizing deletion mutation DE746-A750 in the parental PC-9 cells. However, we failed to detect new cooccurring mutations in the EGFR gene (including the gatekeeper EGFR-T790M mutation, in the tumors generated by the PC-9/ Erl-R cells, which maintained the same expression level of EGFR DE746-A750 as the PC-9 cells ( Supplementary Fig. 1). By using Human EGFR Pathway qBiomarker Somatic Mutation PCR Arrays, we also confirmed that the PC-9/Erl-R tumors did not harbor any secondary mutations in the KRAS, NRAS, or BRAF genes (data not shown).
Oral administration of silibinin delays tumor progression in erlotinib-refractory EGFR-mutant NSCLC mouse xenografts. Silibinin is the primary active constituent of a crude extract (silymarin) from milk thistle plant (Silybum marianum) seeds. We explored the ability of an oral milk thistle extract formulation that was enriched (30% w/w) with a water-soluble form of silibinin complexed with the amino-sugar meglumine to inhibit the growth of a PC-9/Erl-R xenograft animal model. When the tumor volumes reached approximately 100 mm 3 , as measured with calipers, the mice were randomly allocated into groups of 6 animals each. The groups received erlotinib, silibinin, erlotinib and silibinin, or vehicles by oral gavage. The mice in each group were followed, and their tumors were measured during the 35 days of treatment; the tumor volume was calculated for each mouse at each time point. Fig. 2A shows the rate of tumor growth in the four treatment groups, with the data plotted as the mean tumor volume in each group over time. In the erlotinib-treated animals, the tumor volumes were larger than the initial volume at all time points, and this increase became statistically significant at day 14. When compared to the animals in the vehicletreated group (mean tumor volumes of 965 6 155 mm 3 ), 5 weeks of treatment with erlotinib for 5 days/week at 100 mg/kg body weight by oral gavage failed to prevent tumor growth in animals xenografted with PC-9/Erl-R cells, showing a mean tumor volume was as high as 767 6 180 mm 3 . However, 5 weeks of treatment with silibinin for 5 days/week at 100 mg/kg body weight by oral gavage caused a marked time-dependent reduction in xenograft growth. Thus, compared to the mean xenograft tumor volume in both the untreated control and erlotinib-treated animals, the mean tumor volume of the silibinin-treated mice was significantly smaller (460 6 120 mm 3 ). Throughout the duration of the experiment, the mice www.nature.com/scientificreports SCIENTIFIC REPORTS | 3 : 2459 | DOI: 10.1038/srep02459 treated with silibinin did not show any gross signs of toxicity or possible adverse effects, as assessed by body weight gain and diet consumption profiles (data not shown). Taken together, these results demonstrate the in vivo antitumor efficacy of oral silibinin administration on erlotinib-refractory, EGFR-mutant tumor xenografts, without any apparent signs of toxicity.
Systemic silibinin administration sensitizes erlotinib-resistant xenografts to erlotinib. Daily oral gavage of the mice with erlotinib-treated xenografts with silibinin resulted in a dramatic reduction in the mean tumor volume to 143 6 60 mm 3 . Although erlotinib treatment only reduced the tumor volume of the PC-9/Erl-R xenografts by 20% at 35 days post-treatment, the combined treatment of erlotinib and silibinin reduced the tumor volume of the PC-9/Erl-R xenografts by an impressive 85% over the same treatment period. Thus, although the tumors increased to 7 times their initial volume in the group treated with erlotinib, there were no statistically significant changes in tumor size over the same time period in the erlotinib and silibinin-treated mice. These findings strongly suggest that the erlotinib-refractory PC-9/Erl-R xenografts were sensitized to the tumor growth inhibitory effects of erlotinib in the presence of silibinin. This was reflected by the percentage of tumor growth inhibition, which was calculated as 1-treated/control volume ratios (1-T/C) (Fig. 2B). The inhibitory effect of erlotinib was modest, reaching a maximum after 4 weeks (26%) and decreasing toward the end of the treatment to the very low levels of activity achieved during the first weeks of treatment (10%-20%). Conversely, after 2 weeks of treatment, the anti-tumor activity of the single agent silibinin was equivalent to the highest inhibitory effect (24%) of erlotinib, which was achieved after 4 weeks of treatment. The anti-tumor effect of silibinin reached a maximum after 4 weeks (52%) and remained constant until the end of the 5week treatment schedule (52%). Of note, the anti-tumor activity in the combined erlotinib and silibinin group (57%) was already greater after only 7 days of treatment than the maximum activity achieved at any time with the single-agent treatment. Moreover, the inhibitory effect of the combination increased in a time-dependent manner, reaching a maximum of 85% after 35 days.
Oral silibinin reduces the expression of EMT-related markers in erlotinib-refractory tumors in vivo. MicroRNAs (miRNAs) are single-stranded, 19 to 25-nucleotide-long, short RNAs that elicit regulatory effects on the post-transcriptional regulation of genes by binding to the 39 untranslated region (39UTR) of a target messenger RNA (mRNA), leading to translational repression or target mRNA cleavage. It is well established that miRNAs regulate EMT through the regulation of E-cadherin and other molecules, such as the transcription factor ZEB [35][36][37][38][39][40] . Additionally, recent studies have suggested that natural agents, including the polyphenols curcumin, (-)-epigallocatechin-3-gallate, and resveratrol, can alter miRNA expression profiles, leading to the reversal of EMT and enhancing the efficacy of conventional cancer therapeutics [41][42][43][44][45][46][47][48][49][50] . As such, we hypothesized that the plant polyphenol silibinin may also function as a previously unrecognized regulator of miRNAs that might lead to the reversal of the EMT phenotype. After 35 days of oral treatment with silibinin, tumors were collected and snap frozen for the isolation of total RNA. We then used a customized array for the quantitative, real-time PCR (qRT-PCR) analysis of 12 miRNAs that are functionally associated with EMT and the reciprocal mesenchymalto-epithelial transition (MET), namely miR-31, miR-21, miR-10b, miR-103, miR-9, let-7a, miR-200a, miR-205, miR-200c, miR-141, miR-155, and miR-429. Using a cutoff of a 4-fold or greater difference to determine significant regulatory effects on miRNA expression levels, we found that the expression of EMT-driving miR-21 [51][52][53][54][55][56] was drastically augmented in the PC-9/Erl-R-derived tumor tissues (15 6 3-fold up-regulation; Fig. 3). miR-31, another oncogenic miRNA that is closely related to the pro-EMT effects of miR-21 51,57-59 , was also found to be significantly overexpressed in the PC-9/Erl-R-derived tumor tissues (5.7 6 1-fold up-regulation; Fig. 3). Conversely, miR-200c, a member of the miR-200 family and a key regulator of EMT that controls the transition between non-stem cell-like and cancer stem cell (CSC)-like cellular states and that has been shown to determine the response to EGFR TKIs 37,41,60-64 , was found to be significantly underexpressed in the PC-9/Erl-R-derived tumor tissues (23.4 6 1-fold downregulation; Fig. 3). Remarkably, the PC-9/Erl-R tumor samples treated with oral silibinin exhibited a full reversion of the respective up-or down-regulation of miR-21, miR-31, and miR-200c that was observed in the erlotinib-refractory PC-9/Erl-R tumors. Thus, the systemic administration of silibinin was able to restore the altered expression pattern of EMT-related miRNAs, returning them to the control levels observed in the erlotinibresponsive PC-9 parental tumors (Fig. 3).
Recent studies have revealed that miR-21 expression confers tumor aggressiveness and metastatic potential, at least in part, by inducing the expression of and cooperating with the EMT master regulatory gene, SNAIL1 55 . Accordingly, we used qRT-PCR to explore whether the ability of silibinin to down-regulate miR-21 expression affected the expression of SNAIL1. We confirmed that SNAIL1 expression was markedly reduced in the tumor tissues harvested from the erlotinib-refractory PC-9/Erl-R xenografts and that silibinin treatment restored SNAIL1 expression to the basal levels observed in the erlotinib-responsive PC-9-formed xenograft tissues (Fig. 3). Because all the studies published to date have described the epithelial transcriptional repressors ZEB1 and ZEB2 as critical targets of miR-200 family members, we examined whether the ability of silibinin to promote the re-expression of miR-200c significantly impacted the expression of its predicted targets, ZEB1/2. qRT-PCR analyses confirmed that the ZEB1 gene was significantly up-regulated in the erlotinib-refractory PC-9/Erl-R xenograft tissues (Fig. 3). In contrast, oral treatment with silibinin down-regulated ZEB1 gene expression to the basal levels observed in the erlotinib-responsive PC-9 xenograft tissues. These results suggested that the antagonism of miR-21 and re-expression of miR-200c induced by silibinin decreased the mRNA expression of mesenchymal phenotype cell biomarkers. We also studied the relative mRNA expression of Ncadherin (CDH2), a mesenchymal cadherin associated with EMT that has consistently been found to be up-regulated in most biopsies obtained from recurrent NSCLC patients showing signs of EMT in response to the induction of erlotinib resistance 65 . In accordance with our other findings, we found that N-cadherin was significantly decreased in the PC-9/Erl-Rd xenograft tissues following treatment with silibinin.
Silibinin reverses the mesenchymal phenotype of erlotinib-refractory PC-9/Erl-R cells in vitro. To confirm that the effects observed with regard to EMT-related erlotinib efficacy in the mice treated with oral silibinin were also due to the direct effect of silibinin on the tumor cells, and not only on the mouse tissue micro-environment, we evaluated whether silibinin alone was sufficient to reactivate an epithelial phenotype in erlotinib-refractory PC-9/Erl-R mesenchymal cells in vitro. Microscopically, the PC-9/Erl-R cells exhibited a spindle cell-like morphology that was notably different from the parental PC-9 cells. As expected, the erlotinib-refractory PC-9/Erl-R cells displayed EMT features, including the reorganization of filamentous actin (F-actin), with a significant enhancement of stress fibers, a decrease in the abundance of the epithelial cell-cell adhesion protein, E-cadherin, and an increase in the abundance of the mesenchymal adhesion protein vimentin when compared to the erlotinib-responsive PC-9 parental epithelial cells (Figs. 4 and 5). Of note, treatment with silibinin-meglumine induced the re-expression of E-cadherin, which correlated with morphological changes consistent with the erlotinib-refractory PC-9/Erl-R cells undergoing the reciprocal mesenchymal-to-epithelial transition (MET). Analyses of the actin stress fibers and vimentin expression before and after treatment with silibinin-meglumine revealed that the PC-9/ Erl-R cells had reverted from the erlotinib-refractory, acquired mesenchymal state to the original, erlotinib-sensitive, de novo epithelial cellular state (Figs. 4 and 5). Importantly, all these phenomena related to changes in the cell migratory behavior of PC-9/Erl-R cells. In vitro scratch wound healing assays showed that the highly migratogenic phenotype of PC-9/Erl-R cells was largely prevented in the presence of silibinin; thus, silibinin-treated PC-9/Erl-R cells demonstrated a significantly slower cell migration speed compared to the basal locomotory activity of untreated PC-9/ ErlR cells (Fig. 5).

Discussion
By culturing erlotinib-hypersensitive PC-9 cells harboring the erlotinib-sensitizing EGFR exon 19 mutation DE746-A750 in the presence of high concentrations of erlotinib over a period of several months, we were able to isolate PC-9 derivatives capable of growing at erlotinib concentrations of up to 10 mmol/L 34 . We recently described that, in the absence of second-site EGFR mutations, alternative activation of MET, AXL, or HER2, gain of secondary mutations in the KRAS, NRAS, or BRAF genes, and loss of the mutant delE746-A750 EGFR gene itself 66 , acquired resistance to erlotinib is mostly correlated with a significant enrichment in EMT features 34 . By taking advantage of this unique model of non-mutational, EMTdriven acquired resistance to erlotinib, we explored whether an oral formulation of a milk thistle extract enriched (30% w/w) with a water-soluble derivative of the bioactive lignan silibinin (i.e., silibinin complexed with the excipient amino-sugar meglumine) was able to circumvent erlotinib resistance in EGFR-mutant PC-9/Erl-R xenografts. This paper reports, for the first time, the inhibitory effect of silibinin on NSCLC cancer growth in an in vivo xenograft model of EMT-driven acquired resistance to erlotinib that was generated by the subcutaneous injection of PC-9/Erl-R cells in athymic mice. Specifically, we demonstrated that 5 weeks of oral administration of 100 mg/kg per day silibinin was sufficient to significantly inhibit the growth of PC-9/Erl-R tumor xenografts, Importantly, our data demonstrate that silibinin can reverse the resistance to erlotinib because mice concurrently exposed to silibinin and erlotinib show an impressive 85% reduction in tumor growth after 35 days of combined treatment. No evidence of tumor regrowth was noted in the combination group, even after discontinuation of the drugs for two weeks. Although these findings suggest that the biological activity of silibinin might continue after administration has ceased, long-term monitoring of tumor growth and spread in the mice should be performed before unambiguously concluding that silibinin improves recurrence free survival in our erlotinib-refractory xenograft models.  The process of EMT is regulated by the expression status of specific miRNAs during tumor development and disease progression; miR-21 is one such miRNA that is overexpressed in solid tumors compared to paired benign and normal tissues. Indeed, miR-21 is considered to be an oncogenic miRNA that exhibits anti-apoptotic activity in various types of cancer cells and appears to control the EMT process and the CSC phenotype by targeting PTEN or collaborating with the EMT transcription factor SNAIL 51-56 . This suggests  that the antagonism of miR-21 could be therapeutically beneficial for the treatment of drug-refractory tumors that are driven by EMT-like phenomena. The miR-200 family is another example of a group of miRNAs associated with cancer recurrence and overall survival that plays an important role in the regulation of EMT. The low expression of members of the miR-200 family plays an important role in cancer metastasis by controlling the EMT process and EMT-driven resistance to cancer therapies 37,41,[60][61][62][63][64][67][68][69] . The reactivation of miR-200 by novel approaches may serve as an inhibitor of EMT, which may either reverse or eliminate the EMT phenotype, enabling anti-cancer drugs, such as EGFR-directed therapy, to effectively circumvent EMT-associated drug resistance. In this study, we first compared the expression of EMT-related miRNAs between erlotinib-sensitive and erlotinib-refractory NSCLC tumor tissues and investigated whether the natural agent silibinin could function as a novel microRNA-modulating anticancer agent. We found that the expression levels of miR-21 and miR-200c were significantly up-regulated and down-regulated, respectively, in the erlotinib-refractory tumors. Accordingly, these tumors showed EMT characteristics, including a high expression of such mesenchymal markers as SNAIL, ZEB, and N-cadherin. Interestingly, we also documented that the increased expression of miR-21 and attenuated expression of miR-200c, a signature of tumor aggressiveness, in the erlotinib-resistant tumors was significantly affected by treatment with silibinin. Indeed, silibinin treatment down-regulated miR-21 and up-regulated miR-200c, and, consequently, the reversion of the high miR-21/low miR-200c signature resulted in the down-regulation of SLUG, ZEB1, and Ncadherin, a result that was consistent with the reversal of the EMT phenotype. We also showed that the combination of silibinin with erlotinib was much more effective than either agent alone, suggesting that the alterations in specific miRNAs mediated by silibinin, which led to the reversal of the mesenchymal phenotype, could be a novel approach for the prevention of erlotinib resistance in NSCLC treatment. Nevertheless, it is reasonable to suggest that silibinin should be added to the current list of microRNA-modulating anticancer agents that can efficiently target the EMT phenotype.
Our results should prompt further interest in the use of silibinin as a drug modality, as they raise the possibility that the EMT arising during erlotinib-resistance development, as observed in EGFRmutant NSCLC, can be countered by the silibinin combination. This can be readily tested in a clinical setting; indeed, there has been an effort to translate the relevance of preclinical anticancer efficacy studies into the clinical setting, specifically with regard to determining the comparable doses required for humans. In this respect, the calculation of the human equivalent dose (HED) using normalization of the body surface area rather than weight has been advocated 70 . On the basis of the formula proposed for calculating HED, animal dose (mg/kg) x (animal Km factor/human Km factor), we found that the corresponding HED for the maximum dose of silibinin used in our in vivo study, which was equivalent to 100 mg/kg mouse body weight, is a human equivalent dose of 8.11 mg/kg. This equates to a 486.49-mg dose of silibinin for a 60-kg individual, an HED that is likely within the dose that can be achieved in target cancer tissues. When colorectal cancer patients were given 1.45 g/day of silipide, a formulation of silibinin with better bioavailability, for 7 days, this dosage was found to be safe and achieved levels of up to 4 mmol/L in the plasma, 2.5 nmol/g tissue in the liver, and 141 nmol/g in the colorectal tissue 71 . Thus, the effective doses of silibinin used in the present in vivo study are within the range of physiologically achievable concentrations of silibinin found in the tissues of cancer patients given similar doses of silibinin. Moreover, it is known that silibinin has poor water solubility. However, the ionization ability of silibinin increases when it is combined with meglumine, and silibinin-meglumine has a high water solubility. Our development of a water-soluble formulation of silibinin is crucial for target preparations, as it will enable new formulation breakthroughs for silibinin-meglumine-containing injections, granules, or beverages. In addition, one of the principles of combination therapy is that the side effects of the two drugs should not overlap. Considering the relatively non-toxic nature of such natural agents as silibinin, which has been widely used for many years with negligible toxicity, better treatment outcomes may be achieved through the novel and safe approach of targeting EMT-type,  erlotinib-refractory NSCLC cells with silibinin-meglumine combined with EGFR TKIs (such as erlotinib). A highly efficacious, long-term drug-release delivery system for silibinin-meglumine that must be safe after oral or intravenous administration needs to be developed. The pre-clinical findings of the present study, along with the lack of any apparent toxicity associated with silibinin, underscore the efficacy of silibinin against erlotinib-refractory, EGFR-mutant NSCLC, with translational potential in the immediate future.
Because mesenchymal-type NSCLC cancer cells are more resistant to EGFR TKIs than epithelial-type NSCLC cancer cells, regardless of the EGFR mutation status, and given that various erlotinib-resistance mechanisms occur via the induction of EMT, the discovery of pharmacological approaches that are able to reverse the tumor status of EMT features could identify new strategies for sensitizing this important subgroup of EGFR TKI-resistant NSCLC tumors. The results presented in this article showed, for the first time, that the nontoxic, natural agent silibinin can effectively reverse EMT. Specifically, silibinin activates the MET genetic program in EGFRmutant NSCLC cells that have acquired resistance to erlotinib. Mechanistically, this phenotype is associated with the EMT-related high miR-21/low miR-200c microRNA signature (Fig. 6). We also found that silibinin down-regulates the expression of miR-21 and promotes the re-expression of miR-200c in erlotinib-refractory tumors, providing a molecular mechanism for the observed reversal of EMT to MET, along with the complete reversal of erlotinib resistance in vivo. These novel findings suggest that the anticancer activity of the bioactive lignan silibinin against aggressive NSCLC tumors is, in part, due to the reversal of the EMT phenotype. The administration of a water-soluble, silibinin-rich milk thistle extract should be tested in upcoming clinical trials to assess whether it can prevent or reverse EMT-driven NSCLC progression following erlotinib treatment.

Methods
Drugs and chemicals. The water-soluble milk thistle extract was provided by Monteloeder, S. L. in its silibinin-meglumine salt form (Elche, Alicante, Spain). The milk thistle extract composition was analyzed and quantified by HPLC-145 DAD-ESI-MS/MS on an Agilent LC 1100 series (Agilent Technologies, Inc., Palo Alto, CA, USA). The retention time, UV spectra, and MS/MS data of the sample peaks were compared to data reported in the literature [72][73][74] . Quantitation of the main flavonolignan compounds was performed using ChemStation for LC 3D software (Agilent Technologies Life Sciences and Chemical Analysis, Waldbronn, Germany), and the silibinin equivalents were determined with silibinin standards (Sigma-Aldrich). The EGFR (HER1) TKI erlotinib (TarcevaH) was a kind gift from Roche Pharmaceuticals. Stock solutions of erlotinib at 10 mmol/L were prepared in DMSO, and aliquots were stored in the dark at 220uC until use. For the experiments, all drug solutions were prepared fresh from stock solutions and were diluted with the cell growth medium. The control cells were cultured in media containing identical concentrations (v/v) of vehicle.
Generation of PC-9 cells with acquired resistance to erlotinib. PC-9 NSCLCderived cells expressing the EGFR exon 19 deletion mutation (delE746-A750) were obtained from the IBL cell bank (Gunma, Japan). PC-9 cells with acquired resistance to erlotinib were generated as previously described 34 .
Tumor cell implantation experiments. A total of 40 female athymic nude mice (four to five weeks old, 23 to 25 g) purchased from Harlam Laboratories (France) were subcutaneously inoculated with 5 3 10 6 /100 ml PC-9/Erl-R cells. Two independent experiments were performed, and the data obtained from the two experiments were pooled. Subcutaneous tumor growth was monitored daily; when the solid tumors reached a volume of 100 mm 3 , the animals were randomly assigned to groups of 6 animals each. These groups were treated with vehicle control, erlotinib (100 mg/kg, 5 days a week), silibinin-meglumine (100 mg/kg, 5 days a week), or erlotinib plus silibinin-meglumine. All the drugs were administered via oral gavage. The mice were weighed once per week after dosing, the tumor sizes were measured daily with electronic calipers, and the tumor volumes were calculated with the following formula: volume (mm 3 ) 5 length 3 width2 3 0.5. The experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Institut d'Investigació Biomèdica de Bellvitge (IDIBELL; Animal Use Protocol #6302 authorized by the Animal Experimental Commission of the Catalan Government, Barcelona, Spain). After 35 days of oral treatment, the mice were killed by cervical dislocation, and the tumors were collected. The tumor samples were divided for DNA, microRNA, and mRNA analyses.
Quantitative real-time polymerase chain reaction (qRT-PCR). For the purification of microRNA and total RNA from the tumor tissues, we used Qiagen miRNeasy Mini kits (Cat. No. 217004) and QIAshredder columns, according to the manufacturer's instructions. To complete the miRNA PCR assay procedure, we reverse transcribed our small RNA samples into first-strand cDNA, the template for PCR, using the RT 2 miRNA First Strand Kit (Cat. No. MA-03/331401; SABiosciences). Real-time PCRbased miRNA expression profiling was performed with a customized miRNA PCR array in a 96-well format, as described in the SABiosciences RT miRNA qPCR Assays manual. One microgram of total RNA was reverse-transcribed to cDNA using the Reaction Ready TM First Strand cDNA Synthesis Kit (SABiosciences) and applied to a customized PCR array in a 96-well format. The PCR array contained the following panel of genes: GSC, KRT14, KRT19, NUMB, TCF3, TCF4, SDC1, ZO-1, CD44, TWIST, SNAI1, VIM, SLUG, CDH1, ZEB1, CDH2, ZEB2, FN1, and CD24. The arrays were processed according to the SABiosciences RT-PCR manual and analyzed using an Applied Biosystems 7500 Fast Real-Time PCR System with an automated baseline and threshold cycle detection. The data were interpreted using a web-based PCR array analysis tool from SABiosciences.
Immunofluorescence staining and high-content confocal imaging. For imaging, the cells were seeded at a density of approximately 5,000 cells/well in 96-well clearbottom tissue culture plates (Becton Dickinson Biosciences; San Jose, CA) optimized for automated imaging applications. TritonH X-100 permeabilization and blocking, primary antibody staining, secondary antibody staining using Alexa FluorH 488/594 goat anti-rabbit/mouse IgGs (Invitrogen-Molecular Probes, Eugene, Oregon), and counterstaining (using Hoechst 33258; Invitrogen) were performed according to protocols from BD Biosciences. The images were captured in different channels for Alexa FluorH 488 (pseudocolored green), Alexa FluorH 594 (pseudocolored red), and Hoechst 33258 (pseudocolored blue) using a BD Pathway TM 855 Bioimager System (Becton Dickinson Biosciences, San Jose, California) with 203 or 403 objectives (NA 075 Olympus). Merged images were obtained according to the recommended Assay Procedure using the BD Attovision TM software.
Wound-healing motility assays. PC-9/Erl-R cells were seeded onto six-well dishes at 1 3 10 5 per well. A single scratch wound was creased using a p10 micropipette tip into confluent cells. Cells were washed twice with PBS to remove cell debris, supplemented with regular growth medium in the absence or presence of silibinin, and monitored. Images were captured by phase-contrast microscopy at 0, 24, and 48 h after wounding.


extfieldsofstudy: Biology Medicine

text:
GABAergic responses of mammalian ependymal cells in the central canal neurogenic niche of the postnatal spinal cord☆

Highlights • Extensive dye-coupling occurs between mammalian spinal cord ependymal cells.• GABA depolarised all spinal cord ependymal cells tested.• GABA effects were mediated by GABAA receptors but not GABA uptake transporters.

Introduction
The functional characteristics of ependymal cells surrounding the central canal (CC) of the mammalian spinal cord are currently poorly understood. Given the growing belief that the CC region is a postnatal neurogenic niche and that ependymal cells could be acting as neural stem/progenitor cells [2,5] it is important to understand the basic characteristics of ependymal cells and how they respond to changes in their environment. A recent study has made the first steps towards characterising ependymal cells using electrophysiology in the rat, demonstrating that they have a low input resistance (124 ± 24 M ), a resting membrane potential of −84 ± 2 mV and passive response properties to current pulses [16]. Dye-coupling was also observed between these cells and immunohistochemistry revealed the presence of connexin 43 indicating that gap junction coupling occurs between ependymal cells [16]. Gap junction coupling is known to influence proliferation in the developing CNS and is also observed between neural progenitor cells in the postnatal Subventricular zone (SVZ) [3,9]; it could be performing a similar function here.
There are currently no studies investigating the effect of neurotransmitters on ependymal cells surrounding the CC of the mammalian spinal cord. A recent study by Reali et al. [20], explored the effects of GABA on brain lipid binding protein (BLBP) immunoreactive progenitor cells that are found surrounding the CC of the turtle spinal cord; these cells appear to be similar to the radial ependymal cells or potential radial glia that surround the mammalian CC [16,17]. GABA depolarised these progenitor cells inducing increases in intracellular Ca 2+ , suggesting that GABA is capable of stimulating long lasting intracellular effects within these cells [20]. The response to GABA was mediated by either GABA A receptors, the GABA uptake transporter, GAT3, or a combination of both [20], highlighting variation within the population of progenitor cells. Neural stem and progenitor cells within other mammalian postnatal neurogenic niches such as the subventricular zone (SVZ) or the dentate gyrus (DG) also respond to GABA and as a result GABA is capable of influencing the proliferation and differentiation of these cells [12,23].
Given this conserved ability of neural stem and progenitor cells to be able to respond to GABA and the presence of GABAergic terminals forming synapses with ependymal cells surrounding the mammalian CC [14], it is highly likely that GABA is affecting these cells. As ependymal cells are an endogenous source of new cells within the postnatal mammalian spinal cord, it is pertinent to increase both our knowledge about how mammalian ependymal cells communicate between themselves and whether they can respond to external stimuli, such as GABA. This study used whole cell patch clamp electrophysiology and intracellular dye-loading to demonstrate that ependymal cells in the postnatal spinal cord were capable of responding to GABA and that this response was in part mediated by GABA A receptors.

Animals
Wistar rats (P9-21) were used in line with the Animals Scientific Act (1986) and the ethical standards set out by the University of Leeds Ethical Review Committee by individuals with Home Office approval. Every effort was made to minimise the number of animals used and their suffering.

Whole cell patch clamp electrophysiology
Recordings were made at room temperature. Viable ependymal cells were viewed at 60× magnification using differential interference contrast (DIC) imaging and QCapture Pro software via a camera (QImaging Rolera-XR, Q imaging, Scientifica, UK) attached to the microscope (Olympus BX50W1). Whole cell patch clamp recordings were made with microelectrodes filled with intracellular solution (mM): K gluconate (110), EGTA (11), MgCl 2 (2), CaCl 2 (0.1), HEPES (10), Na 2 ATP (2), NaGTP (0.3). Neurobiotin (0.5%; Vector Laboratories, USA) and tetramethylrodamine (0.02%; Invitrogen, USA) were added to allow visualisation of the neurone post-recording. E Cl calculated using the Nernst equation from the stated solutions was −103 mV. Current clamp recordings (to enable characterisation of the cells) were performed using an Axopatch-1D amplifier (Molecular Devices) and signals were captured by pClamp9 software (Molecular Devices). The membrane potential measurements were not corrected for the liquid junction potential. To characterise voltage responses, depolarising and hyperpolarising current pulses of 1 s duration (ranging from −50 pA to +50 pA) were applied. The input resistance was determined by the size of the hyperpolarisation in response to the injection of −50 pA current pulses.

Drugs
All drugs were prepared as stock solutions in water unless otherwise stated and bath applied using a gravity perfusion system. GABA (200 M; Sigma-Aldrich) was bath applied alone or coapplied with bicuculline methochloride (100 M, Ascent Scientific, UK), gabazine (2.5 M, dissolved in DMSO, final concentration 0.1%, Sigma-Aldrich), guvacine hydrochloride (50 M, Tocris Bioscience, UK) or ±nipecotic acid (300-900 M, Tocris Bioscience, UK). (R) baclofen (10 M, Tocris Bioscience, UK) was applied alone to determine whether cells responded to activation of GABA B receptors. 18␤-glycyrrhetinic acid (18␤-GA) potassium salt (100 M, Sigma-Aldrich) was bath applied to investigate the presence of gap junctions. On several occasions, GABA (500 M) was pressure ejected using a PV800 pneumatic picopump (World Precision Instruments Ltd., UK, 10 psi; 500 ms pressure duration) from a separate micropipette placed in the vicinity of the recorded cell.

Analysis
Changes in membrane potential and input resistance were recorded before, during and after drug applications and all data expressed as mean ± standard error (S.E.). Drug effects were determined using paired t-tests or one-way ANOVAs with post hoc Bonferroni tests and differences between drug effects determined using independent t-tests.

Morphological identification of ependymal cells
Slices were imaged at the end of recordings using epifluorescence with a filter of 555 nm. The recording electrode was removed and the slice fixed in 4% PFA + 0.25% glutaraldehyde for detailed post hoc visualisation. Slices were washed in 0.1 M phosphate buffer, gelatine embedded and resectioned into 50 m sections using a vibrating microtome. Sections were permeabilised in phosphate buffered saline (PBS) containing 0.1% triton X-100 before being incubated in extraavidin peroxidase (1:250 in PBS) for 36-72 h then washed in 0.1 M PBS. Cells were visualised by incubating in diaminobenzidine (DAB; 0.5 mg/ml in tris buffer + 1% H 2 O 2 ) until the staining was visible.

Ependymal cells in the mammalian spinal cord have passive response properties and are coupled by gap junctions
The ependymal cells recorded had a resting membrane potential of −76 ± 0.5 mV (n = 79), small input resistances (96 ± 13 M ; n = 79) and displayed no spontaneous or evoked activity (Fig. 1A). There was a linear relationship between the change in voltage in response to a current pulse and the size of the current pulse (Fig. 1Ai); this demonstrates that ependymal cells have passive responses indicative of a lack of voltage-gated ion channels. A Spearman's rank order correlation test found a significant but weak positive correlation between the resting membrane potential and the input resistance (r s (79) = 0.337, P = 0.002), demonstrating that ependymal cells with more negative resting membrane potentials generally have smaller input resistances. Cells were confirmed as ependymal cells by a lack of processes when visualised by rhodamine or Neurobiotin post-recording (n = 79; Fig. 1Aii).
Neurobiotin, which was present within the intracellular solution, is gap junction permeable and allows coupling between cells to be observed [24]. The visualisation of Neurobiotin with DAB revealed that the ependymal cells were highly coupled (n = 28 out of 28 recovered for Neurobiotin; Fig. 1B). There was variation in the size of the dye-coupled clusters, with some clusters covering almost the whole CC region (Fig. 1Bi). Post-recording visualisation of cells with rhodamine, which is not gap junction permeable [7], showed no coupling of cells, indicating that there was no spillage of tracers. To confirm that the transfer of dye was through gap junctions, the non-selective gap junction blocker 18␤-GA (100 M; [6]) was bath applied. 18␤-GA increased the input resistance of ependymal cells by an average of 360 ± 201 M (n = 6; Fig. 1C), with increases ranging from 57 M to 1346 M . A Wilcoxon Signed Rank Test determined that this increase was significant (Z = −2.201, P = 0.028). The range of changes in input resistance suggests that the ependymal cells are gap junction coupled to different degrees, supporting the dye-coupled data.18␤-GA also depolarised ependymal cells to different degrees (0-52 mV). A Spearman's Rank Order correlation found a strong, significant correlation between the change in input resistance and the change in membrane potential (r s (4) = 1.000, P < 0.01; Fig. 1Ci). This suggests that the depolarisation that occurs in response to the application of 18␤-GA is related to blockade of gap junctions and not a non-gap junction related effect of 18␤-GA.   Fig. 2Ai and B). However, the size of this depolarisation was significantly smaller than that in GABA alone (t (3) = 3.685, P = 0.035), with GABAergic responses in bicuculline being 38 ± 10% (n = 7) of the initial GABA response. Bath application of gabazine (2.5 M) also antagonised GABA responses, with a 37 ± 11% (n = 2) decrease in the size of the depolarisation. The effects of both bicuculline and gabazine were reversible (Fig. 2B).

Ependymal cells of the mammalian spinal cord respond to GABA
It was investigated whether the remainder of the GABAergic response could be mediated by GABA transporters, as observed in ependymal cells of turtle spinal cord [20]. The bath application of GABA in the presence of nipecotic acid (900 M) or guvacine (50 M), non-selective GABA transporter blockers, resulted in a significant depolarisation of ependymal cells (4.11 ± 0.67 mV; n = 7; Fig. 2C). The size of this depolarisation was not significantly different (P = 0.34) from that of ependymal cells responding to GABA under control conditions (4.17 ± 0.67 mV; n = 7; Fig. 2 Ai and C). Furthermore, co-application of either guvacine or nipecotic acid and bicuculline still failed to completely block the response mediated by bath application of GABA (1.55 ± 0.63 mV; n = 3; Fig. 2Ai and C).

Discussion
This study provides an electrophysiological characterisation of ependymal cells surrounding the CC and is the first study to demonstrate that ependymal cells in this area within the postnatal mammalian spinal cord respond to GABA. Ependymal cells displayed typical characteristics of glial cells, with no spontaneous or evoked activity, indicating a lack of voltage-gated channels. Dye coupling with Neurobiotin following intracellular loading confirmed reports that ependymal cells are coupled and the gap junction blocker 18␤-glycrrhetinic acid established that this coupling was mediated by gap junctions. Ependymal cells consistently depolarised to GABA, an effect partially antagonised by GABA A receptor antagonists, bicuculline and gabazine, but the remainder of the response was not decreased by GABA transporter blockers, nor was the response mimicked by the GABA B agonist baclofen. The ability of these cells, which are considered to be neural stem cells, to respond to GABA is extremely pertinent and highlights the need for further studies investigating how GABA affects the proliferation and differentiation of these cells.
The input resistance of 96 M in ependymal cells is slightly lower than that previously determined for ependymal cells in the rat spinal cord, 124 M [16]. As connexin expression is known to increase steadily from P0 to adulthood in other CNS areas [10], the lower input resistance here may be due to the older animals used (P11-P21) compared to that of Marichal et al. ([16] P0-P5). The lack of spontaneous or evoked activity and the linear voltage-current relationship agrees with previous studies of rat and turtle spinal cord ependymal cells [15,16,21] and suggests that ependymal cells lack voltage-gated ion channels.

The relevance of gap junction coupling
This study confirmed previous reports that gap junction coupling occurs between ependymal cells of the rat spinal cord [16]. As 18␤-GA is a non-selective gap junction blocker, the specific identity of the connexin subunits forming the gap junctions was not identified, however, immunohistochemistry implies that either connexin 43 [16,19] and/or connexin 45 [4] form the gap junctions between ependymal cells. The strong correlation between the change in input resistance and the change in membrane potential in response to 18␤-GA indicates that the depolarisation is a direct effect of gap junction blockade rather than a non-gap junction specific effect of 18␤-GA. This effect is similar to that observed in progenitor cells surrounding the turtle CC [20]. A possible reason for ependymal cells to form gap junctions is to allow the control of cellular proliferation, as seen in the embryonic neocortex and in the adult SVZ [3,11]

Could GABA influence ependymal cells?
The depolarisation of ependymal cells observed following bath or focal application of GABA resembles that observed in progenitor cells surrounding the CC of the turtle spinal cord [20] and in the postnatal neurogenic niches of the brain [12,23]. Given that E GABA is predominantly influenced by E Cl − , which was −103 mV in this study, a hyperpolarisation rather than a depolarisation would have been expected. Although the presence of the Na + -K + -2Cl − co-transporter (NKCC1) in ependymal cells would not generally be enough to overcome the low intracellular Cl − concentration imposed by the intracellular solution within the patch pipette, if the NKCC1 channels were expressed in close proximity to GABA A receptors in the cell membrane, a local accumulation of intracellular Cl − could explain the depolarisation. The high degree of gap junction coupling could also allow the movement of Cl − into the recorded cell, however, this is unlikely to be sufficient to raise intracellular Cl − concentration. Most likely, the depolarisation resulted from an intense activation of GABA receptors, as commonly observed [18,22]. This prolonged activation of GABA A receptors can lead to an imbalance of HCO 3− efflux and Cl − influx that shifts E GABA away from E Cl − towards E HCO 3-, which was −12 mV here. A final speculation was that the depolarisation could in part be a result of a contribution by GABA uptake transporters which transport GABA back into the cells with two Na + ions and one Cl − ion, thus carrying a net positive charge into the cell [13]. The lack of effect of nipecotic acid or guvacine, non-selective GABA uptake transporter blockers, in this study suggests that this is not the case. It also highlights a difference between the ependymal cells of the turtle spinal cord, where GABA transporters contributed to the GABAergic response and those investigated here in the rat spinal cord. Other similarities were observed between the two species, including the presence of GABA A receptors mediating the GABAergic response. However, in rats it remains to be determined what is mediating the bicuculline resistant component of the GABAergic response.
The endogenous source of GABA is likely to be the GABAergic terminals that synapse with ependymal cells [14]. These terminals could originate from either local GABAergic interneurons or from neighbouring GABAergic cerebrospinal fluid contacting neurones (CSFcNs) that are in the subependymal layer of the CC [1]. The CSFcNs may release GABA into the CSF from their CSF-contacting processes, enabling widespread distribution of GABA to ependymal cells.
The fact that ependymal cells have properties of neural stem cells [2,8,25] and GABA influences the proliferation and differentiation of neural stem cells in other neurogenic niches [12,23] suggests that GABA could be influencing the proliferation and/or the differentiation of ependymal cells surrounding the CC. In the postnatal neurogenic niches of the brain, GABA appears to reduce the proliferation of neural stem/progenitor cells and induce differentiation to produce more newborn neurones [12,23]. Unlike the postnatal neurogenic niches of the mammalian brain and lower vertebrate spinal cord, ependymal cells undergo only symmetrical division to maintain the ependymal cell population under physiological conditions [8]. If cells within the CC area respond to GABA in a similar way to the SVZ and DG, the proliferation of ependymal cells rather than differentiation suggests a lack of endogenous GABA under physiological conditions. It is possible that following an injury or the onset of a pathological condition, GABA could be released around this area, limiting proliferation and promoting differentiation. If this is the case, being able to manipulate this GABAergic modulation would enable a greater control over the neurogenic capacity of this area.

Conclusion
This study demonstrates that ependymal cells surrounding the CC of the postnatal mammalian spinal cord are capable of responding to the neurotransmitter GABA. Ependymal cells could be more integrated into the spinal cord circuitry than previously expected and may be capable of responding to changes in the environment, with potential consequences for the neurogenic capacity of the area.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Surface-Enhanced Raman Spectroscopy (SERS) Tracking of Chelerythrine, a Na+/K+ Pump Inhibitor, into Cytosol and Plasma Membrane Fractions of Human Lens Epithelial Cell Cultures

Background/Aims: The quaternary benzo-phenanthridine alkaloid (QBA) chelerythrine (CET) is a pro-apoptotic drug and Na+/K+ pump (NKP) inhibitor in human lens epithelial cells (HLECs). In order to obtain further insight into the mechanism of NKP inhibition by CET, its sub-cellular distribution was quantified in cytosolic and membrane fractions of HLEC cultures by surface-enhanced Raman spectroscopy (SERS). Methods: Silver nanoparticles (AgNPs) prepared by the Creighton method were concentrated, and size-selected using a one-step tangential flow filtration approach. HLECs cultures were exposed to 50 μM CET in 300 mOsM phosphate-buffered NaCl for 30 min. A variety of cytosolic extracts, crude and purified membranes, prepared in lysing solutions in the presence and absence of a non-ionic detergent, were incubated with AgNPs and subjected to SERS analysis. Determinations of CET were based on a linear calibration plot of the integrated CET SERS intensity at its 659 cm-1 marker band as a function of CET concentration. Results: SERS detected chemically unaltered CET in both cytosol and plasma membrane fractions. Normalized for protein, the CET content was some 100 fold higher in the crude and purified plasma membrane fraction than in the soluble cytosolic extract. The total free CET concentration in the cytosol, free of membranes or containing detergent-solubilized membrane material, approached that of the incubation medium of HLECs. Conclusion: Given a negative membrane potential of HLECs the data suggest, but do not prove, that CET may traverse the plasma membrane as a positively charged monomer (CET+) accumulating near or above passive equilibrium distribution. These findings may contribute to a recently proposed hypothesis that CET binds to and inhibits the NKP through its cytosolic aspect.

Introduction
Chelerythrine (CET) is a quaternary benzo-phenanthridine alkaloid (QBA) of 348 Daltons mass and extracted from Papaveraceae, i.e. plants of the Poppy family. In solutions near physiological pH, CET is positively charged (CET + ) due to an iminium group (N + =C) attracting small anions such as Cl. At increased pH values, CET + loses its charge and becomes a pseudobase with an apparent pK a value of ~9 [1] . In the past, QBAs such as CET and its sister alkaloids, the sanguinarines, have been widely used as medicinal remedies in insecticides, bactericides, fungicides, viricides, in ointments, mouthwash solutions, and tooth pastes, sedatives and anti-helmintics [2]. More recently, CET had been first reported as a protein kinase C (PKC) inhibitor [3] and, utilizing this mechanism, later heralded as a potential anticancer agent capable of inducing apoptosis in a variety of cancer cell lines [4][5][6]. Acting as a BH3-mimetic, CET induces apoptosis by binding to BH1-like motifs of B cell lymphoma type 2 (Bcl-2) proteins inhibiting pro-survival B-cell lymphoma extra large (BclXI) proteins and activating mitochondrial Bcl-2 associated X (Bax)/ Bcl-2 homologous antagonist-killer (BAK) protein pro-apoptotic mechanisms [7][8][9].
Recently, CET was shown to inhibit ouabain-sensitive K + or Na + /K + pump (NKP) influx in human lens epithelial cells (HLECs) without interference with ouabain binding and PKCmediated NKP phosphorylation [10]. The CET structure-related sanguinarines have been known for a long time to inhibit the NKP in a variety of tissue preparations [11][12][13][14][15][16][17], without an explanation for this effect. Sequence homologies between BH1-like motifs originally reported to bind CET in Bcl-Xl proteins and found in the cytoplasmic aspect of the crystal structure of the NKP led to the hypothesis that CET may inhibit the NKP function through these BH1 motifs [10]. The amino acid sequence ARAAEILARDGPN of the first putative CET binding site resides in the A domain of the NKP and has two arginine (R) residues. Interestingly, fluorescence studies suggest interaction of R residues with sanguinarines, however, in the carboxy (C)-terminus of the NKP [18]. Moreover, sequence homologies of the NKP BH1-like motifs found in all P-type ATPases suggest other cellular metabolic pathways can be influenced by the presence of CET [10].
Although QBAs have been shown to rapidly diffuse across plasma membranes and may be used as fluorescing DNA probes [19], the mechanism of transmembrane distribution of CET is unknown and thus the question as to whether the charged monomeric CET + or its lipid soluble pseudobase traverse the plasma membrane prior to binding and affecting intracellular structures such as the NKP remains unresolved. Traditional techniques for monitoring the amount and localization of active metabolites in biological systems (i.e., ultraviolet-visible absorption spectrophotometry, fluorescence spectroscopy, liquid chromatography, etc.) are often limited by low detection limits, increased experimental duration and cost, or indirect observation of the species of interest via a (radio) chemical "label". Recently, surface-enhanced Raman spectroscopy (SERS) has emerged as a powerful molecular sensing technique due to its high degree of molecular specificity, low detection limits, and multiplex detection capabilities [20,21], permitting accurate determination of distribution and amount of metabolites in biological systems with very short experimental durations, a conditio sine qua non for pharmacological studies.
In order to obtain insight into the interaction of CET with the NKP, this study attempts to track the distribution of CET across the plasma membrane into the cytosol of HLECs, our model, in which CET was shown to inhibit the NKP [10]. Raman analysis of a stock CET solution exhibited a few very weak peaks; however, upon the addition of silver nanoparticles (AgNPs), a large signal enhancement was observed yielding vibration-rich spectra. The SERS enhancement of CET facilitated the construction of a calibration function for determination of the concentration of monomeric CET in the cytosol and its content in membrane particulate fractions. The alkaloid CET accumulated in the cytosol and plasma membranes at concentrations near or above diffusion equilibrium suggesting transport as an organic cation CET + , presumably the species interacting with Bcl-2 proteins and NKP [10].

Cell Culture and Preparation of Cytosol and Membrane Extracts
HLECs were obtained as a primary fetal human lens cell line (FHL124) by courtesy of Dr. John Reddan, Oakland University, Michigan, and were cultured and propagated using specialty support and media as described earlier by this laboratory [10,22,23]. Cells were grown to near confluence in 100 mm diameter Petri dishes (49.1 cm 2 surface area/dish), culture media aspirated, washed twice with each 4 mL room temperature (RT)-equilibrated balanced salt solution (BSS), and exposed to 4 mL phosphate buffered saline (PBS) without or with 50 μM CET in the presence and absence of dimethylsulfoxide (DMSO) at 37 o C for 30 min, a time sufficient to nearly completely inhibit NKP [10]. Thereafter, cells were washed thrice with 4 mL ice-cold PBS, all supernatant fluids removed, and each dish covered with 0.4 mL M-PER ® (Pierce Biochemicals) lysing solution containing 1:100 HALT reagent to stop proteolysis, and maintained for 10 min on a shaker at RT. Cellular debris was collected into Eppendorf tubes, which were then spun for 2 min at 14,000 g in an Eppendorf RT centrifuge to separate the cytosolic supernatants from the crude membrane particulate material. It was noted that the cytosol of the CET-treated cultures was slightly yellow, suggesting that CET had entered the cells. The crude membrane pellet was resuspended and washed 4 times in fresh M-PER ® solution, after which the final pellet was dissolved in 0.5 mL M-PER ® solution containing 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), a non-ionic detergent known to solubilize membrane proteins by replacing membrane lipids. These final solubilized crude membrane samples were thrice ultra-sonicated on ice with 3 bursts to achieve maximal membrane disintegration. All samples were spun at 14,000 g for 2 min, the supernatant kept and the small finite pellet discarded. Aliquots of 0.05 mL supernatant were removed for protein analysis by the BIORAD® technique. Samples were stored at -20 o C for further SERS-based analysis.
In order to obtain a purified membrane preparation, HLECs, after CET exposure, were washed as described above, but then collected from the Petri dishes by scraping into a HALT-containing NKP Na +conformational buffer. The scrape was sonicated on ice as described above, and then centrifuged in a Beckman Optima XL ultracentrifuge (Beckman Co, Stanford Ca) at 4,600 g for 10 min at 4 o C to remove mitochondria and nuclear material followed by a final centrifugation at 131,000 g for 1 h at 4 o C to obtain the final plasma membrane pellet. This membrane pellet, further solubilized by sonication in NKP buffer containing 1% CHAPS and stored in aliquots at -80 o C, possesses only the α1 subunit of the NKP [10].

Estimation of Cell Volume and Calculation of Cytosolic Dilution
Based on previous estimates of endothelial and lens epithelial cell volume [23,24] of 10 -11 L/cell and an average cell count of 10 6 /dish, a total cell volume of 10 -2 mL/dish was estimated. As an example, given that 3 Petri dishes/control or experimental cells were available, which received a total M-PER lysing solution of 1.2 mL, the final total volume was 1.23 mL, i.e., the dilution of the original cytoplasm was estimated to be 41 fold; and 71 in another experiment.

AgNP Synthesis, Size-Selection, and Concentration
AgNPs were synthesized in a batch-wise process via a modified Creighton method [25] employing the ice-cold reduction of AgNO 3 (1.0 mM, 50 mL) by NaBH 4 (2.0 mM, 300 mL). The formation of spherical AgNPs of moderate size distribution (~1-200 nm in diameter [26] was confirmed by the presence of an intense, symmetric ultraviolet-visible (UV-VIS) absorption peak at 392 nm. Once characterized, individual batches of AgNPs (350 mL each) were combined to achieve a total volume of 4.0 L. Tangential flow filtration [27] was then employed to size-select AgNPs, while eliminating the reaction byproducts/excess reagents and increasing the concentration of nanosilver for optimum SERS-based sensing. It was recently shown that the optimal size of AgNPs for SERS enhancements is ~50 nm in diameter [28]. Briefly, 4.0 L of colloid was recirculated through a hollow fiber polysulfone filter module (50 nm pore size, 390 cm 2 surface area, Spectrum Labs®) yielding a retentate of AgNPs of increased diameter (≥ 50 nm) and a filtrate containing dilute, smaller AgNPs (< 50 nm). The concentrated AgNPs (Ag 50C ) were stored at ~ 4 °C until further use in SERS measurements.

Raman and SERS Measurements and Analysis
Raman and SERS measurements were conducted using a LabRamHR 800 system (Horiba Scientific, Inc.) coupled to either an internal HeNe laser (632.8 nm, 15 mW) or an external, frequency-doubled Nd:YAG laser (532.1 nm, 15 mW) with an acquisition time of 20 s and 3 s, respectively. The laser beam was focused into a 2 mL quartz cuvette carrying the Raman or SERS sample via a high stability, confocal microscope (BX 41) using a 50X objective. Serial dilutions of a CET stock solution (20 mM) were performed prior to the SERS analysis, yielding concentrations of 10 -3 M through 10 -9 M. Aliquots of the CET solutions were briefly incubated with the Ag 50C colloid, followed by addition of 1.0 M KBr to induce AgNP aggregation and SERS "hot-spot" formation [28] prior to analysis. SERS samples of HLEC extracts of the plasma membrane and cytosol were prepared in a similar fashion, and the total CET concentration in each extract was estimated via a standard calibration curve constructed from the SERS scattering intensities of the stock CET solutions. Peak integration routines and user-defined baseline correction algorithms were performed in Origin 8.5 software for each Raman and SERS spectrum.

Raman and SERS Analysis of Monomeric CET
Prior to SERS analysis, the UV-VIS absorption spectrum of 10 -4 M CET revealed absorption peaks at 212, 267, 315, 338 and 406-430 nm (Fig. 1A). The extinction spectrum  Figure 1B. CET contains a highly chromophoric imine bond, which experiences pre-resonance effects under 532 nm excitation and leads to a large fluorescent background in the Raman spectrum. However, the Raman spectrum of monomeric CET exhibited weak, yet distinctive vibration modes upon excitation via the 632.8 nm HeNe laser ( Fig. 2A). Characteristic functional group-vibration modes and those reported in the literature were utilized in order to provide a tentative assignment for each peak observed within the Raman spectrum (Table  1). Raman spectra of monomeric CET exhibited low signal-to-noise ratios for concentrations lower than 10 -2 M, i.e., far above the physiologically active concentrations of the monomer. The addition of AgNP 50C to stock solutions of CET not only yielded an exponential increase in scattering intensity, but also effectively quenched the fluorescence resulting from pre-resonance effects at concentrations as high as 10 -4 M. The SERS spectra of monomeric CET showed a vibration-rich structure (Fig. 2B). In addition, the large increase in SERS signal observed for CET-AgNP 50C mixtures yielded sub-picomolar detection limits (i.e, 10 -10 M), which are well below IC 50 values reported in the literature for diverse inhibitory functions by this drug. Vibration-rich SERS spectra (data not shown) were also observed for CET solutions containing the original unmodified AgNP colloid.
However, the SERS spectra using the modified Ag 50C exhibited greater spectral intensities, which is in good agreement with literature reports of SERS scattering as a function of AgNP size. SERS spectral analysis of vibration bands was performed in a similar manner as for the regular Raman scattering spectra (Table 1). It should be noted that the SERS analysis of CET has been sparsely reported and the assignments provided here serve only as tentative descriptors.
The four vibration modes occurring at 430 (o.p. τ(C-C-C)), 527 (o.p. sextant σAryl), 659 (in bold in Table 1, defining stretched (C-C-C) bonds) and 778 cm -1 (o.p. (CH)) were utilized as "marker bands" of CET (Table 1) due to the large spectral intensity and lack of overlap with adjacent peaks. Therefore, the presence of any one of these four marker bands in cellular extracts can confirm the presence of monomeric CET. In addition to determining several characteristic marker bands for spectral identification, the in-plane skeletal stretching mode at 659 cm -1 was observed to have the greatest intensity of all the marker bands and was thus utilized to quantify the concentration of CET in biological samples. The constructed calibration function revealed a linear relationship between the normalized 659 cm -1 peak areas and the concentration of the CET solutions from 10 -5 M to 10 -10 M (Fig. 3). Despite the presence of the four marker modes, accurate peak integration parameters could not be obtained for solutions greater than 10 -5 M due to the increased fluorescence of free CET molecules.
Small aliquots of plasma membrane and cytosolic HLEC extracts (50 -100 µL) were quantitatively diluted with either water (Raman) or the Ag 50C and KBr (SERS) to a total volume of 2 mL prior to spectral analysis. The normal Raman spectrum of the cytosolic extracts showed few, yet distinctive peaks corresponding to the DMSO solvent utilized in the extraction procedure ( Figure 4A). A large fluorescent background was observed for the crude plasma membrane extract under normal Raman scattering conditions (i.e., in the absence of AgNPs), thus obscuring all vibration modes aside from a lone DMSO peak at 679 cm -1 . No CET  (Fig. 4).
The SERS spectra of CET-treated cytosolic, and crude and purified plasma membrane fractions exhibited the four distinct marker bands of CET (Fig. 4B, 5), even when using   relatively short acquisition times (3 sec in each). In addition, the extensive biological matrix present in each sample did not appear to affect the intensity of each marker band. This is likely the result of a high degree of affinity between the positively charged iminium nitrogen and the predominantly negatively charged AgNPs in each sample. Previous SERS analyses of molecules of similar structure containing positively charged amino groups (e.g., rhodamine 6G) further corroborate this finding [28]. Total areas for the 659 cm -1 peak/sample were normalized, and concentrations were interpolated using the empirical relation derived from the calibration procedure and the dilution factors ( Table 2). The data in Table 2 yield the CET molar concentration (column 5) measured in the 20 fold-diluted cytosol and membrane extracts (column 1). Extracts A and B are from the same experiment, and C and D from different experiments. In A/B HLECs were disintegrated by MPER, and only the insoluble crude membrane material further solubilized with 1% CHAPS. It is evident that on a mol CET μg -1 protein basis there was 3x10 -2 more CET in the crude membrane fraction than in the cytosol. Based on the calculated dilution factor of the HLEC cytosol of 41 (see Methods), the [CET] i in the cytosol is computed as 43 x 10 -6 M, which is close to the extracellular [CET] 50 x 10 -6 M. When the crude membranes were subjected to two centrifugation steps (see Methods), the resulting plasma membrane pellet (Table 2 C) exhibited a ~70% reduction in CET consistent with riddance from other particulate material such as endoplasmic reticulum, Golgi and mitochondrial membranes and most likely nuclear material. Still, normalized to protein, most of the CET preferred to lodge within the membranes. For the whole cell extract obtained in the presence of 1% CHAPS to solubilize all membrane material (with exception of a small but distinct CET-positive insoluble pellet), the CET content μg -1 protein was within the same order of magnitude, yielding 1.97 x 10 -11 instead of 2.11 x 10 -11 CET content μg -1 protein, consistent with similar cytosolic concentrations of CET in near equilibrium with the external media.
In conclusion these data suggest that SERS is capable to determine the free cytosolic concentration of monomeric CET, but also shows that a rather large CET membrane pool exists suggesting that the total CET that is associated with the cell probably exceeds diffusion equilibrium.

Discussion
Previously, it was proposed that the QBA CET inhibits NKP by binding to at least one BH1 motif discovered within the NKP's actuator (A) domain, thus, for example constraining the 120 o rotation of the A domain and its temporary protection of the high energy aspartate 369-phosphate residue that confers the conformation required for the trapping of the 3 Na + ions in an occluded state [10].
The rationale for the present study was to provide proof of concept by establishing the distribution of CET between the cytosol and the crude plasma membrane preparation using a novel technological approach, SERS, with the ultimate goal to identify the compound within the NKP structure. The use of the large signal enhancement of SERS over ordinary Raman spectra gave us the advantage to a) characterize CET based on its four marker bands (Fig.  1), b) identify it in concentrations less than 10 4 orders of magnitude smaller than Raman spectroscopy would have done ( Fig. 2A and B) and thus establish a calibration curve (Fig. 3) over five orders of magnitude, and c) identify within cytosol and crude and purified plasma membrane fractions, CET concentrations in the micromolar range ( Fig. 4 and 5, Table 2). Therefore, the greater sensitivity of the SERS method coupled to the minimal pre-resonance effects of the 532 nm laser excitation wavelength also easily surpassed by several orders other approaches such as UV-VIS spectrometry, where the limit of detection was around 10 -4 to 10 -5 M (c.f. Fig. 1B). Furthermore, these results show extreme promise in the ability of SERS to be used as a sensitive and specific technique for tracking small molecule cellular metabolites with visible absorption resonances, even within crude cellular matrices. As shown in Table 2, the CET cytosolic concentrations were quite close to that of the bathing medium of 50 μM, raising the question as to whether CET might equilibrate across the plasma membrane, as a positively charged monomer (CET + ) or as pseudo base that does exist at higher pH values [30]. For the sake of the argument, if CET traversed the membrane as CET + , then, according to the Nernst Equation: log [CET + ] i = log [CET] o -E m /61.5, the free intracellular CET concentration, [CET] i , should have been 5 fold above equilibrium at 244 μM, assuming a membrane potential, E m , of -40 mV typical in epithelial cells, where [CET] o is the external CET concentration = 50 μM, and 61.5 mV = 2.3 RT/zF at 37 °C with the usual meanings. If we assume that the cytosolic and membrane CET content add up altogether within one cellular compartment, the total [CET] i should be 267 μM, a value that is close to that predicted from the Nernst equation. Thus it is not improbable that indeed CET traverses as CET + rapidly equilibrating perhaps through cationic channels that are capable to accommodate the 348 dalton compact benzo-phenanthridine ring. In this context, it is of interest that epithelial cells have at least two isoforms of organic cation channels, OCT1 and OCT2 [32], and their presence is currently evaluated in terms of their capability to transport positively charged QBAs. Moreover, it has also been shown that transport through epithelial OCTs plays a pivotal role in the targeting of positively charged QBAs to the plasma membrane. The time-dependent fate of CET cellular distribution will be further investigated to shed light on this issue.
Finally, based on the calculated number of 10 13 CET molecules μg -1 crude membrane protein and assuming that at least 10% of the membrane protein is NKP with a molecular weight of 10 5 Daltons, one can compute a ratio of CET/NKP of about 100, a number still far exceeding the expected ratio of not more than 2 based on the putative presence of two BH1 motifs/NKP with solvent accessibility for CET. Experiments ongoing with affinity purified NKP using mouse-anti human α1 NKP subunit antibody either from plasma membrane-free cytosolic or from purified membrane fractions suggest a CET/NKP ratio of <1. Differences currently addressed may be due to a) insufficient washing of the crude membrane particles releasing all trapped CET only in the presence of CHAPS, b) the tendency of CET to partition and lodge within the structurally not unrelated cholesterol component of the membrane bilayer, c) charge interactions with negatively charged lipids and proteins with neuraminic acid residues, d) absorption to residual nuclear material in particular in the crude membrane fraction, e) trapping of the alkaloids within putative organic cation channels, and last but not least interferences of solvents and CET-binding macromolecules with AgNP augmentation of the electromagnetic signals, i.e. SERS-based nano-sensing.

Conclusions
The NKP is a target of QBAs and recent findings implicate at least two BH1-like motifs within the crystal structure of the transporter to which CET, a BH3-mimetic, might bind and inhibit function [10]. Using a novel approach, SERS-based nano-sensing, the distribution of the QBA CET between the plasma membrane and the cytosolic extracts of HLECs was studied. The data suggest that CET most likely travels as the charged monomeric CET + , because already the free cytosolic CET concentration approached the external CET concentration, and the plasma membrane contained CET exceeding that of the cytosol on a protein basis by more than 2 orders of magnitude.


s2fieldsofstudy: Biology Medicine

text:
Immunophenotype and differentiation capacity of bone marrow-derived mesenchymal stem cells from CBA/Ca, ICR and Balb/c mice.

AIM
To assess the capacity to isolate and expand mesenchymal stem cells (MSC) from bone marrow of CBA/Ca, ICR and Balb/c mice.

METHODS
Bone marrow of tibia and femur were flushed, cultured and maintained in supplemented Dulbecco's modified Eagle's medium. MSC immunophenotype of cultures were tracked along increasing passages for positivity to CD106, Sca-1 and CD44 and negativity to CD45, CD11b and MHC class II. Differentiation capacity of MSC towards osteogenic and adipogenic lineages were also assessed.

RESULTS
MSC were successfully cultured from bone marrow of all 3 strains, albeit differences in the temporal expression of certain surface antigens. Their differentiation into osteocytes and adipocytes were also observed. MSC from all 3 mouse strains demonstrated a shift from a haematopoietic phenotype (CD106(-)CD45(+)CD11b(+)Sca-1(low)) to typical MSC phenotype (CD106(+)CD45(-)CD11b(-)Sca-1(high)) with increasing passages.

CONCLUSION
Information garnered assists us in the decision of selecting a mouse strain to generate MSC from for downstream experimentation.

marrow of all 3 strains, albeit differences in the temporal expression of certain surface antigens. Their differentiation into osteocytes and adipocytes were also observed. MSC from all 3 mouse strains demonstrated a shift from a haematopoietic phenotype (CD106 -CD45 + CD11b + Sca-1 low ) to typical MSC phenotype (CD106 + CD45 -CD11b

INTRODUCTION
Mesenchymal stem cells (MSC) are a great therapeutic interest for tissue regeneration and immunomodulation. To elucidate the role of MSC in these two paradigms, experimentation is often performed with MSC generated from mouse bone marrow. For this, researchers have a choice to make in selecting the mouse strain to generate their MSC from. Here, we provide a report on the comparative description of mouse MSC derived from 3 different strains under the same experimental conditions. The mouse strains assessed were CBA/Ca, ICR and Balb/c. MSC are stromal cells with self-renewal and differen-

BRIEF ARTICLE
tiation properties [1] . MSC have been isolated from various tissues including bone marrow, adipose, skin, bone, synovial membrane, placenta and umbilical cord [2][3][4] and proliferate in vitro as plastic-adherent cells with fibroblastlike morphology [5] . The long term self-renewal and multilineage differentiation properties of MSC reflect their potential for tissue regeneration and cell/gene therapybased treatment of diseases [6] including acute graft-vs-host disease [7] , osteogenesis imperfecta [8] , cardiomyopathy [9] and Crohn's disease [10] . Recent findings that MSC have immunosuppressive properties [11][12][13][14] have also increased the need for a suitable supply of MSC for experimental research. The expansion of undifferentiated MSC is a research tool for functional and genetic studies for subsequent development of preclinical protocols to treat a wide range of diseases. Murine sources of MSC are of great use for the extensive preclinical studies that are required within this research area. Murine MSC is commonly isolated from the bone marrow aspirates of the femur and tibia. Studies demonstrate that isolation of MSC from murine bone marrow using standard methods usually results in a heterogeneous cell population with a high degree of nonmesenchymal contaminants [2,[15][16][17] . Furthermore, there are reports of preferential growth of MSC from different mouse strains [17] . MSC isolated from four different inbred mouse strains C57Bl/6J, Balb/c, FVB/N and DBA1 showed great variation in growth rate, differentiation capacity and immunophenotype [17] . In this study, we compare phenotypic characteristics of MSC derived from bone marrow of 3 strains of mice (CBA/Ca, ICR and Balb/c) to determine a suitable source for these cells for downstream experimentation. As MSC that are cultured lack specific markers [5] , cells were immunophenotyped for a range of surface markers classically associated with MSC expression. The differentiation capacity of MSC confirmed the multipotency of these stem cells.
Although there were differences in temporal pattern of surface marker expression and preferential differentiation, bone marrow cultures from all 3 strains of mice tested successfully yielded MSC.

Isolation and culture of bone marrow-derived MSC
Bone marrow-derived MSC were generated from 3 different strains of mice, namely CBA/Ca, Balb/c and ICR. Mice used were male and between the ages of 6-10 wk. Animals were sacrificed by a CO2 overdose and bone marrow cells were obtained by flushing femurs and tibias with Dulbecco's modified Eagle's medium (DMEM). Cells were centrifuged at 289 g for 10 min, resuspended and seeded into three 25 cm² flasks in DMEM with high glucose supplemented with 10 nmol/mL GlutaMAX™-I Supplement, 100 U/mL penicillin and 100 μg/mL streptomycin, 1 mL/L gentamycin, 250 μg/mL Fungizone (all Invitrogen), 1.5 g/L sodium bicarbonate and 15% Mesenchymal Stem Cell Stimulatory Supplement (STEM-CELL Technologies, Canada). Cultures were maintained at 37 ℃ in 95% humidified air and 5% CO2. After 48 and 72 h, cells were washed gently using 1X PBS and replaced with fresh culture media. Adherent cells were further cultured with a medium change every 3-4 d. At approximately 80% confluency (occasionally localised), cells were harvested by treating with 0.25% trypsin containing 1 mmol/L EDTA for 5 min at 37 ℃. Trypsin was neutralised with culture medium and detached cells were replated in a new 25 cm 2 culture flask. Cell cultures were routinely assessed using an inverted phase contrast microscope and cell viability was determined via trypan blue staining.

Flow cytometry analysis of cell surface markers
The following antibodies were obtained from Becton Dickinson: fluorescein isothiocyanate-conjugated antimouse CD106 (vascular adhesion molecule-1), CD11b, and MHC Ⅰ; phycoerythrin-conjugated anti-mouse CD45, Sca-1, and MHC Ⅱ and allophycocyanin-conjugated anti-mouse CD44. Cells were trypsinised, washed with 0.1% BSA/PBS and incubated with antibody (1 μL per 10 6 cells) for 30 min at 4 ℃. Cells were then resuspended in 0.1% BSA/PBS and analysed by a FACS Calibur cytometer (BD Biosciences, San Jose, CA) using CellQuest Pro software. Ten thousand gated events were recorded. For each antibody, gating was determined based on appropriate isotype-stained controls.

MSC differentiation
Mouse bone marrow cultures of passages 10-16 were plated at a density of 6 × 10 4 cells/well in a 24-well plate and incubated at 37 ℃ in 95% humidified air, 5% CO2 till confluency was reached. For adipocytic differentiation, the Mesenchymal Stem Cell Adipogenesis Kit (Chemicon; Cat. No. SCR020) was used. Briefly, cells were stimulated with induction medium (DMEM with high glucose supplemented with 10% FBS and 1X penicillin and streptomycin, 1 μmol/L dexamethasone, 0.5 mmol/L IBMX, 10 μg/mL insulin and 50 μmol/L indomethacin) and maintained with maintenance medium (DMEM with high glucose supplemented with 10% FBS and 10 μg/mL insulin) according to kit instructions. For analysis, cells were fixed with 4% paraformaldehyde and stained with 0.36% Oil Red O solution for 50 min. For osteogenic differentiation, the Mesenchymal Stem Cell Osteogenesis Kit (Chemicon; Cat. No. SCR028) was used. Briefly, cover slips were coated with 12 μg/mL vitronectin and 12 μg/mL collagen prior to cell seeding. Cells were then stimulated with induction medium (DMEM with high glucose supplemented with 10% FBS and 1X penicillin and streptomycin, 0.1 μmol/L dexamethasone, 0.2 mmol/L ascorbic acid 2-phosphate, 10 mmol/L glycerol 2-phosphate and 1X glutamine). Cells were replaced with fresh induction medium every 2 d. For analysis, osteocytes were fixed with 70% ethanol and stained with Alizarin Red S. For both differentiation assays, controls were MSC cultures without adipocytic/osteogenic induc-tion medium.

Morphology of bone marrow cell cultures
Bone marrow cells were isolated from the femurs and tibias of a total of 11 mice (4 ICR, 2 CBA/Ca and 5 Balb/c mice) and plated onto culture flasks. Non-adherent cells were removed by replacing media and cultures were ob-served via phase-contrast microscopy for their morphology. Initially, cells of all 3 strains of mice were small and exhibited spindle-shaped morphology ( Figure 1, first and second row). Cells also tended to be locally confluent, growing in distinct colonies (as seen for ICR mice, first row; Balb/c mice, second row). A small number of round and flattened cells were observed within the colonies. As cells approached confluency within culture flasks, they were trypsinised and replated into new flasks. Bone mar-

Immunophenotypic profile of bone marrow cultures
Immunophenotyping of bone marrow cultures derived from the 3 mouse strains were performed at various passages throughout the culture period. Presence of MSC were confirmed by positivity to CD106, CD44, Sca-1 and MHC Ⅰ markers and negativity to CD45, CD11b, and MHC Ⅱ by flow cytometry.
Flow cytometric analyses revealed that MSC demonstrated a shift from a haematopoietic phenotype to typical MSC phenotype with increasing passages. MSC of all 3 strains of mice showed this shift in phenotype from CD106 -CD45 + cells at earlier passages to CD106 + CD45at later passages ( Figure 2). ICR mice maintained their CD106 positivity for longer, remaining > 77% from passage 8 to 19. For CBA/Ca and Balb/c mice, CD106 ex-pression began reverting after passage 17 and passage 18 respectively. Expression of CD45 (a common leukocyte antigen) was strong (70%-99%) at early passages for all 3 strains but decreased with increasing passages to < 9% positivity. Cultures derived from Balb/c mice took longer to lose their CD45 positivity compared to CBA/Ca and ICR strains (Figure 2). Similar to CD45, expression of haematopoietic marker CD11b also revealed a shift from CD11b + to CD11bwith increasing passages (Figure 3). MSC cultures from all 3 strains of mice showed high positivity to CD11b (69%-99%) at early passages. Positivity for CD11b decreased with later passages. Cultures from CBA mice achieved CD11bphenotype earlier than the other strains, beginning from passage 5. For the MSC marker Sca-1, cultures showed lower positivity to Sca-1 at early passages with only 24%-50% cells positive (Figure 3). Sca-1 expression increased to a higher percentage after passage 5 for CBA/Ca mice, passage 6 for ICR mice and passage 9 for Balb/c mice. At late passages, MSC from all 3 mouse strains showed strong positivity for Sca-1 at 84%-100%. ICR mice demonstrated a distinctly positive (96%-100%) population for Sca-1 after passage 6, while expression in CBA/Ca and Balb/c mice were 91%-97% after passage 15 and 84%-99% after passage 12 respectively. Next, CD44, MHC Ⅰ and MHC Ⅱ expression was examined in bone marrow cultures with CD106 + CD45phenotype. For CD44 expression, cells from ICR and Balb/c mice had the highest expression (77%-99% and 79%-99% accordingly), compared to CBA/Ca mice that had varying CD44 expression ranging from 13%-90% ( Figure 4). All 3 strains of mice consistently demonstrated positivity to MHC Ⅰ but not MHC Ⅱ (< 2%) (Figure 4).
Results from MSC immunophenotyping shown above suggest that bone marrow cultures from all 3 strains of mice acquire an MSC phenotype (CD106 + CD44 + Sca-1 + MHCⅠ + CD45 -CD11b -MHCⅡ -) after multiple rounds of passaging. Also, amongst the 3 mouse strains tested, bone marrow cells for ICR and Balb/c maintained MSC phenotype for longer compared to CBA/Ca.

Differentiation assay
Bone marrow cultures from all 3 strains of mice differentiated into adipocytes and osteocytes when stimulated with adipocytic and osteogenic induction medium respectively ( Figure 5). Three weeks of exposure to an adipocytic induction medium resulted in formation of lipid vacuoles in cultured MSC that could be observed with phase-contrast microscope. When cultured MSC were exposed to osteogenic induction medium, they aggregated and formed calcium deposits after 2 wk. Both of these cell differentiations were confirmed with cell-specific stains -Oil Red O for adipocytes and Alizarin Red S for osteocytes. Oil Red O-stained lipid vacuoles appeared bright red whereas Alizarin Red S stained precipitated calcium deposits dark red ( Figure 5).

DISCUSSION
Isolation of mouse MSC is generally more difficult than human and rat MSC due to a high number of contaminating haematopoietic lineage cells and slow cell growth [2,15,17] . Studies have also demonstrated mouse strain-dependent variability of MSC in terms of growth kinetics, surface markers and potential for differentiation [16][17][18] . With the protocol described here for bone marrow-derived MSC expansion, we provide descriptive data that MSC can be successfully generated from 3 different mouse strains, namely CBA/Ca, ICR and Balb/c. Bone marrow cells were cultured and each culture was characterised for their morphology, immunophenotype and differentiation potential. MSC from all strains tested showed expression of relevant surface markers and differentiation potential into osteogenic and adipocytic cells, albeit some differences in terms of time to achieve MSC immunophenotype.
Isolation of MSC from mouse bone marrow usually results in a highly heterogenous cell population with a high degree of haematopoietic contaminants such as macrophages and fibroblasts [2,16] . Immunophenotyping analysis revealed early passages of our bone marrow cultures to be positive for haematopoietic markers CD45 and CD11b (a macrophage-specific marker). Continuous change of cell culture medium removes non-adherent haematopoietic populations from the cultures [3] . Passaging bone marrow cultures also helps eliminate haematopoietic contaminants and yield purer MSC cultures [17] . With these approaches, we accordingly observed the MSC cultures become increasingly homogenous at later passages with minimal CD45 expression profile by P7-P8, although it took a little longer for cells from Balb/c mice. Better quality MSC cultures were also obtained by dis- carding cells that remained attached to flasks following trypsinisation. These firmly adherent cells are probably contaminating fibroblasts and have been reported by other laboratories [17,19] . Expression of stem cell marker Sca-1 also increased at later passages. Sca-1 is a marker associated with MSC and also haematopoietic and endothelial progenitors [20] . MSC also expressed positivity to CD44 and CD106 and negativity to MHC Ⅱ. To further encourage MSC growth and colony formation, we used 15% of a mouse mesenchymal supplement (MesenCult ® , STEMCELL Technologies) instead of the routine 10% FBS to obtain enriched MSC cultures.
In addition to morphology and phenotype, the characterisation of MSC is complete with demonstration of their capacity for multilineage mesenchymal differentiation [15,18,19,21] . All 3 mouse strains exhibited multilineage potential in vitro, differentiating into adipocytes and osteocyte when stimulated with appropriate induction media. MSC from Balb/c mice more readily differentiate into adipocytes than the other strains, also shown by Peister and colleagues who found Balb/c more readily differentiating into adipose cells than C57Bl/6J and DBA 1 mice [17] . Conversely, MSC from Balb/c took longer to differentiate into osteocytes compared to the other 2 strains tested in our study. In view of the duration that MSC cultures remained in suitable phenotype and their ease to differentiation, our group has primarily utilised ICR mice for downstream experimentation where we show these stem cells to have immunomodulatory properties [13] .
MSC from all 3 different strains of mice tested were suitable sources for bone marrow-derived MSC as they showed typical morphology, immunophenotype and differentiation capacities of MSC. However, bone marrow cultures from Balb/c mice took longer than the other strains to achieve MSC immunophenotype.

Background
Experimental research concerning mesenchymal stem cells (MSC) relies heavily on cells isolated from animals. In this paper, we sought to determine whether MSC could be successfully generated from three different mouse strains. The ICR, CBA/Ca and Balb/c mice strains were available to us in the laboratory, and are strains commonly used in research. However, there is no literature stating the feasibility of each of these strains to yield bone marrow-derived MSC. It is important to determine the strain to use, as strain-dependent variability has already been reported by Peister and colleagues.

Research frontiers
The therapeutic relevance for MSC is not limited to their regenerative properties, but also their capacity for modulation of immune responses. Beyond the efficacy of MSC to ameliorate disease, it is essential to test the immunogenicity and tumorigenicity of these cells. The use of murine-derived MSC for preclinical research is indisputable and their applicability in experimental settings should be determined for researchers to be able to accurately assess their use.

Innovations and breakthroughs
In this article, the authors show that bone marrow MSC can be derived from ICR, CBA/Ca and Balb/c mice strains with the protocol described. Similar information for these three mouse strains was previously unavailable. With passaging, bone marrow cultures achieved MSC immunophenotype and displayed multipotency by differentiating into osteocytes and adipocytes.

Applications
This study demonstrates that ICR, CBA/Ca and Balb/c mice strains can yield bone marrow-derived MSC for downstream experimentation. Based on the protocol described for culture of MSC, the authors describe the passage range of bone marrow cultures for each mouse strain that yields MSC (based on immunophenotyping data). This information provides data that can assist researchers faced with a choice of setting up mouse bone marrow-derived MSC cultures.

Peer review
This article assessed bone marrow-derived MSC cultivated among three different mouse strains. The authors found some differences between strains with regards to the expression of certain surface antigens and differentiation capacity. These results will be useful for the selection of mouse for preclinical studies concerning bone marrow MSC.