s2fieldsofstudy: Biology Medicine

text:
Cancer metastasis through the prism of epithelial‐to‐mesenchymal transition in circulating tumor cells

Metastasis of epithelial cancer cells to distant sites is a particularly critical stage of cancer progression that typically marks the incurability of the disease. It is governed by a complex series of events including invasion and intravasation of tumor cells into the stroma and blood, respectively. Epithelial‐to‐mesenchymal transition (EMT), a phenotypic change marked by the loss of epithelial characteristics and the acquisition of invasive mesenchymal properties, is implicated in the dissemination of tumor cells. Circulating tumor cells (CTCs), the precursors of metastasis, can be used to interrogate the contribution of EMT in metastasis and therapeutic responses. The analysis of these CTCs and in particular the presence of inter‐ and intrapatient heterogeneity for markers of EMT has provided new insights into the metastatic process. This review will focus on epithelial–mesenchymal plasticity in CTCs and its potential clinical implications.

Introduction
Epithelial-to-mesenchymal transition (EMT) is a tightly regulated lineage change occurring during gastrulation, neural crest delamination, and heart valve formation, developmental processes involved in embryonic patterning (Savagner, 2015). Hallmarks of EMT include the loss of epithelial markers and the acquisition of mesenchymal markers, disruption of cell-cell junctions, and apico-basal polarity as well as remodeling of the cytoskeleton. These changes are associated with increased cell migration and resistance to anoikis (Savagner, 2015) and are triggered by several growth factors, cytokines, and numerous transcription factors. EMT is a reversible process that is dependent on the continued presence of the EMT-inducing signal, the removal of which results in the reversion of the mesenchymal cell to an epithelial state [mesenchymalto-epithelial transition (MET)].
In adult organisms, EMT is activated during physiologic and pathophysiologic responses including wound healing and fibrosis (Stone et al., 2016). EMT has been extensively implicated in cancer progression, specifically in promoting the early stages of metastasis, which involve invasion of tumor cells into the surrounding stroma and blood-borne dissemination of cells to the lung, liver, bone, and brain (Nieto et al., 2016;Thiery et al., 2009). Once epithelial cancer cells, which have transitioned to a mesenchymal state, reach distant sites, it is postulated that they revert to the epithelial lineage through the process of MET (Gunasinghe et al., 2012). Tumor cells that are able to survive within the new microenvironment can acquire the ability to proliferate and establish metastatic tumors, which in most cancers marks a stage in which the cancer becomes incurable. In addition to a role in tumor cell invasion and dissemination, EMT of epithelial tumor cells is also linked to the acquisition of stem-like characteristics and drug resistance (Aktas et al., 2009;Arumugam et al., 2009;Jolly et al., 2015;Marchini et al., 2013;Sequist et al., 2011;Witta et al., 2006). Interestingly, lineage tracing of epithelial and mesenchymal tumor cells within genetically engineered mice shows that EMT may not be a prerequisite for metastasis, but instead contributes to drug resistance, suggesting additional functions for EMT in cancer progression (Fischer et al., 2015;Maheswaran and Haber, 2015;Zheng et al., 2015).
Cancer cells co-opting EMT to migrate and invade provides an attractive model to understand the critical steps involved in the initiation of metastasis, a highly complex process. However, identifying, within the primary tumor, epithelial tumor cells converting to a mesenchymal state has been complicated by the presence of stromal cells which express high levels of mesenchymal markers. As such, despite the dramatic invasive and tumorigenic phenotypes observed in mouse xenografts expressing master EMT-inducing transcriptional regulators, Snail, Twist, and Slug among others (Ocana et al., 2012;Tran et al., 2014;Yang et al., 2004;Ye et al., 2015), the direct observation of EMT in the metastasis of human epithelial cancers has remained elusive. Increased invasion of tumor cells undergoing EMT suggests that mesenchymal characteristics would be prevalent in tumor cells entering the circulation. EMT of tumor cells in the blood has been evaluated by PCR analysis of peripheral blood mononuclear cells isolated from patients with cancer (Aktas et al., 2009;Lasa et al., 2013), but this approach has been unreliable due to the high rate of false signals detected in unpurified blood components. Analysis of circulating tumor cells (CTCs), precursors of metastasis which circulate in the blood as either individual tumor cells or as tumor cell clusters/tumor emboli, offers a rare opportunity to gain insight into the spread of cancer at a critical stage of metastasis (Maheswaran and Haber, 2010). Accumulating evidence from the analysis of these isolated CTCs has demonstrated significant heterogeneity of EMT markers supporting the concept of EMT as an important feature of invasive cancer cells (Fig. 1).
Recent advances in technology have revolutionized our ability to isolate and characterize CTCs from the blood of patients with numerous forms of cancer (Ferreira et al., 2016). The development of these innovative CTC technologies has lead to the concept of using CTCs as a liquid biopsy that may be able to provide prognostic information upon initial diagnosis, track response to therapies, and identify early signs of resistance to treatment (Lianidou et al., 2015). Additionally, the analysis of CTCs that are derived from different sites of disease within a patient with metastatic disease has the potential to better detect the intrapatient heterogeneity of the cancer cells compared to a site-directed biopsy (Fig. 2). The study of CTCs has revealed that markers of EMT are an important feature that can help define and characterize these cells and provide valuable and potentially actionable information to clinicians regarding disease prognosis and response to therapy for individual patients (Satelli et al., 2016;Yu et al., 2013;Zhao et al., 2017). This review will focus on the current state of knowledge regarding EMT in circulating tumor cells.

Biophysical and functional property-based enrichment of CTCs
CTCs were first observed in the blood of patients with cancer almost 150 years ago (Ashworth, 1869). They represent a rare population of cells, which are estimated to be present at a frequency of one CTC admixed with a billion blood cells (Maheswaran and Haber, 2010). Therefore, their isolation presents a significant technological challenge. Given their clinical utility as noninvasive blood-based biomarkers, several platforms have been developed to enrich CTCs from the blood of patients with cancer. Some approaches rely on the functional properties of CTCs including the ability to breakdown collagen and invade or secrete detectable proteins (Deng et al., 2014;Lu et al., 2010). Other approaches use physical characteristics of the tumor cells including size, density, electric charge, and deformability to distinguish them from blood cells   Hou et al., 2013;Liu et al., 2015;Mitchell et al., 2015;Shaw Bagnall et al., 2015). Each method must be evaluated independently to determine the ability to enrich for tumor cells with either epithelial or mesenchymal features (Table 1). For example, there is little difference in the deformability between epithelial and mesenchymal tumor cells, suggesting that CTC enrichment relying on deformability might represent an unbiased approach to capture both CTC lineages (Shaw Bagnall et al., 2015). EMT, however, is associated with an increase in cell size (Lamouille and Derynck, 2007), suggesting that size-based CTC separation methods might be biased toward the enrichment of mesenchymal CTC populations. In fact, size-based CTC collection from patients with different types of metastatic cancers shows enrichment of CTCs with mesenchymal and stem-like characteristics (Hou et al., 2013;Wu et al., 2015). This may reflect increased tumor aggressiveness, but needs to be considered with the caveat that size-based CTC separation is biased toward enrichment of larger cells.

Antibody-based CTC isolation
The most common approach underlying CTC isolation technologies is dependent on immunoaffinity purification of tumor cells. In one form of this method, antibodies are directed against proteins that are exclusively expressed on the plasma membrane of the tumor cells and not on hematologic cells resulting in the positive selection of CTCs. Alternatively, antibodies against proteins that are expressed on the surface of the white blood cells can be used to mark and deplete the normal blood components-negative depletion-and the CTCs remaining in the flow-through can be collected for further analysis.

Positive selection of CTCs
Numerous technologies relying on positive selection of CTCs utilize antibodies against EpCAM, a cell surface glycoprotein expressed on tumor cells of epithelial lineage, but not on leukocytes. CellSearch, the only FDA-approved CTC isolation device, is based on immunomagnetic separation of CTCs expressing EpCAM and enumeration of the captured cells by staining with a cocktail of antibodies against the epithelial cytokeratins 8, 18, and/or 19 (Andree et al., 2016). CellSearch identifies CTCs in about 50% of patients with metastatic disease, which may represent the technical limitation of the technology, or the variable expression of the epithelial markers (Allard et al., 2004). Enumeration of CTCs using this technology is prognostic in patients presenting with many types of epithelial cancers, with the presence of ≥5 CTCs per 7.5 mL of blood being predictive of poor clinical outcome (de Bono et al., 2008;Hayes et al., 2006). CTC technologies including CellSearch, which rely on EpCAM-mediated capture of CTCs, are limited by their inability to enrich mesenchymal CTCs which either express low levels or no EpCAM. CTCs that fail to be captured by EpCAM-dependent methods are detectable with antibodies directed against mesenchymal as well as other tumor markers (Raimondi et al., 2011;Schneck et al., 2015), suggesting that CTCs are indeed heterogeneous for epithelial and mesenchymal markers. To overcome the limitation of EpCAMmediated CTC enrichment and to capture CTCs irrespective of their EMT status, antibody cocktails against EGFR and HER2 (tumor-specific markers), CDH11 (a cadherin specifically overexpressed in mesenchymal tumor cells), as well as cell surface vimentin (expressed in cancer cells of mesenchymal lineage)  (Armstrong et al., 2011;Satelli et al., 2015a;Yu et al., 2013), have been used in combination with EpCAM antibodies. Importantly, broadening the spectrum of detection antibodies beyond those solely against epithelial cytokeratins has improved the identification of mesenchymal CTCs in the blood (Armstrong et al., 2011;Satelli et al., 2015a;Yu et al., 2013).

Negative depletion of leukocytes
The efficiency of positive selection technologies is significantly impeded by the extensive heterogeneity exhibited by tumor cells in the blood. The negative depletion approach based on the removal of normal blood components circumvents this problem leading to antigenindependent purification of CTCs. This minimizes the loss of CTC populations, whose heterogeneity can be attributed to various determinants including EMT. As highly specific antibodies with high affinity against mouse leukocyte populations are readily available, the negative selection approach can also be readily applied to isolate CTCs from mouse tumor models, negating the search for mouse-specific antibodies required for positive selection of these CTCs (Ting et al., 2014). However, this approach may also enrich nonhematopoietic cells such as circulating endothelial cells as well as other circulating epithelial cells (Goon et al., 2006). Thus, CTC identification and enumeration following negative depletion requires carefully chosen epithelial and mesenchymal markers benchmarked against healthy donor blood, as well as molecular analyses that ensure their tumoral origin (Yu et al., 2013). Nevertheless, staining CTCs with epithelial and mesenchymal markers following negative depletion shows that CTCs in the blood are comprised of both epithelial and mesenchymal tumor cells (Yu et al., 2013).

EMT of epithelial cancers
In addition to the significant advances in the ability to isolate CTCs, a second important advance in the study of CTCs is the development of analytical tools including single-cell RNA-Seq (Saliba et al., 2014;Tang et al., 2010), multicolor RNA in situ hybridization (Wu et al., 2016), and single-cell DNA methylation analysis available to characterize CTCs (Pixberg et al., 2017). While still in the development stages, the potential rapid advancement of single-cell proteomics will likely also prove to be a critical tool (Macaulay et al., 2017). The single or clustered nature of CTCs and their rarity necessitates the use of these technologies; however, the analysis of CTCs with these technologies has provided important insight into the biology of CTCs including their heterogeneity. Future technologic advancements will permit the multimodality analysis of the same single cell to give a richer view of CTC biology.
RNA in situ hybridization (RNA-ISH) using multiple probes to mark epithelial and mesenchymal states has been applied to CTCs to increase detection sensitivity without compromising specificity (Yu et al., 2013). A dual colorimetric RNA-ISH using two pools of probes, one against seven epithelial markers (keratins (KRT) 5,7,8,18,and 19;EpCAM, and the other against three mesenchymal markers [(FN1 (fibronectin 1), CDH2 (cadherin 2), and SERPINE1/PAI1 (serpin peptidase inhibitor, clade E)], was used to visualize breast CTCs isolated by positive selection using antibodies against HER2, EGFR, and EpCAM. Semiquantitative measurement of the punctate epithelial and mesenchymal RNA-ISH signals can define the degree of epithelial and mesenchymal characteristics of each individual CTC. These results showed that CTCs exhibit varying degrees of epithelial and mesenchymal characteristics (e.g., epithelial (E), mesenchymal (M), E > M, E = M, and M > E), demonstrating that EMT is a continuous process. In fact, the biphenotypic tumor cells simultaneously expressing both epithelial and mesenchymal markers may represent the most plastic and potentially the cells most likely to contribute to metastatic outgrowth (Jolly et al., 2015). The EMT status of CTCs in patients with breast cancer is dependent on the breast cancer subtype: Mesenchymal CTCs are prevalent in triple-negative and HER2-positive breast cancer patients, whereas CTCs in lobular breast cancer patients are predominantly epithelial (Yu et al., 2013). Lobular breast carcinoma is marked by mutations or loss of E-cadherin, a critical epithelial protein. Yet, tumor cells within the primary tumor or metastatic lesion do not exhibit complete conversion to a mesenchymal state although these tumors are marked by an invasive growth pattern (McCart Reed et al., 2016). Analysis of tumor cells invading through the blood is consistent with these findings. Further studies are needed to continue to define what elements of epithelial plasticity contribute to metastatic spread and potentially the cooperative interactions between cells with different phenotypes.
In addition to breast cancer, heterogeneity of EMT markers in CTCs has also been observed in other cancers. RNA-ISH analysis of CTCs isolated from patients with liver, nasopharyngeal, breast, colon, gastric, and non-small-cell lung cancer showed the presence of epithelial, biphenotypic, and mesenchymal populations . The ratio of mesenchymal CTCs increased based on the TNM stage of the cancer for all of the cancers analyzed . A separate study of a mouse model of pancreatic cancer also identified varying degree of EMT in CTCs (Rhim et al., 2012). Interestingly, cells exhibiting mesenchymal markers are also seen in premalignant pancreatic intraepithelial neoplasms and in circulating pancreatic-derived cells prior to the development of overt cancer. Therefore, EMT and malignant cell dissemination may occur early in the development of cancer. Additionally, while tumor cells displaying mesenchymal markers are not significantly enriched in the CTCs, functional analysis revealed that these cells had increased tumor-initiating potential. Together, these results support the concept of epithelial and mesenchymal heterogeneity in CTCs and provide evidence that EMT may not only permit invasion and escape from the primary tumor, but may induce increased tumor-initiating properties.
Analysis of epithelial and mesenchymal CTC populations in a cohort of patients with breast cancer showed a dynamic shift in the populations during treatment, such that increasing number of mesenchymal CTCs correlated with treatment failure and disease progression (Yu et al., 2013). Increase in the mesenchymal CTC population during relapse is associated with the appearance of tumor emboli or multicellular CTC clusters in the blood. The CTC clusters are highly mesenchymal and are not readily detectable with antibodies against epithelial cytokeratins. The mesenchymal state of the CTC clusters may be attributed to the coating of platelets, which are a rich source of TGF-b, a potent inducer of EMT (Labelle et al., 2011). Indeed, transcriptome analysis of the breast CTC clusters shows the enrichment of TGF-b and EMT signatures, suggesting that CTC clusters, which are oligoclonal and held together through cell-cell interactions, most likely arise through collective migration of tumor cells, a process dependent on partial EMT Friedl and Gilmour, 2009;Yu et al., 2013).
RNA sequencing of single CTCs has also enabled the comprehensive analysis of these cells and has been used to interrogate the EMT characteristics of CTCs in mouse tumor models as well as in patients. The microfluidic device, the iChip, which removes red blood cells and platelets through size-based separation, enriches CTCs by magnetophoretic depletion of immunomagnetically tagged white blood cells (Karabacak et al., 2014). These CTCs collected in solution can be picked as single cells using a micromanipulator and analyzed with RNA-Seq at single-cell resolution Ting et al., 2014). This approach was applied to CTCs isolated from a genetically engineered mouse model of pancreatic cancer [(LSL-Kras G12D , Trp53 flox/flox or + , PDX-Cre (KPC)], which closely mimics the progression of human pancreatic cancer. Single-cell RNA-Seq performed on individual CTCs identified three distinct populations: CTCs expressing epithelial markers (classical CTCs-CTC-c), CTCs enriched for platelet markers (CTC-plt) and CTCs with a robust proliferation signature (CTC-Pro) (Ting et al., 2014). The classical CTCs, in comparison with tumor cells within the primary tumor, exhibited a universal loss of E-cadherin, demonstrating their propensity to lose epithelial markers. This is consistent with the concept of EMT contributing to the invasion of these mouse pancreatic tumor cells into the stroma and blood (Rhim et al., 2012). However, the expression of mesenchymal genes in these CTCs is more complex and highly variable. When compared to the primary tumor, the expression of some EMT markers, CDH11 and vimentin, is elevated in CTCs, whereas others, S100A4, Itga5, Sdc1, are decreased. The heterogeneity exhibited by CTCs in these mice is consistent with the continuity and complexity associated with the EMT process and the presence of several intermediate EMT states in CTCs in human cancer patients. The transition of epithelial cancer cells to a mesenchymal state has been shown to be closely associated with the acquisition of stem-like characteristics (Guo et al., 2012;Mani et al., 2008). Interestingly, the mouse pancreatic CTCs expressed high levels of Aldh1a1 and Aldh1a2 mRNA, but the expression of these stemness markers did not correlate with EMT, suggesting that these phenotypes are not linked in pancreatic CTCs. The absence of a correlation between the mesenchymal and stemness markers is also observed in CTCs isolated from castration-resistant prostate cancer patients .
A transgenic mouse model of prostate cancer tumorigenesis and metastasis was paired with a vimentin-GFP reporter to track the expression of this mesenchymal marker in prostate tumor cells (Ruscetti et al., 2015). Analysis of the peripheral blood of these mice at different stages of tumorigenesis revealed an increase in mesenchymal (EpCAMÀ/GFP+) and biphenotypic (EpCAM+/GFP+) CTCs correlating with metastatic disease. Interestingly, while the mesenchymal and biphenotypic CTCs have increased tumorinitiating ability in the prostate, only the biphenotypic and epithelial CTCs could form macrometastases. The mesenchymal CTCs persisted as micrometastatic foci. These results support the idea that epithelial plasticity is critical to metastasis. However, it is important to point out that in a study of prostate cancer CTCs derived from patients with metastatic disease, RNA-Seq data of single cells did not identify downregulation of epithelial markers or upregulation of mesenchymal markers in subpopulations of the CTCs , suggesting either that the heterogeneity can be lost in a moderately pretreated cohort or that epithelial and mesenchymal heterogeneity is not well represented in single-cell RNA-Seq analysis. Further studies are needed to investigate the presence of epithelial and mesenchymal marker heterogeneity in patient-derived prostate samples.

EMT signatures in CTCs from nonepithelial tumors
Besides epithelial tumors, enrichment of EMT signatures is also observed in CTCs derived from nonepithelial tumor types, including melanoma and glioblastoma. Loss of E-cadherin is observed in late-stage malignant melanoma (Alexaki et al., 2010;Miller and Mihm, 2006) and the EMT-inducing transcription factor network significantly changes during melanomagenesis and constitutes a risk factor for poor outcome in malignant melanoma (Caramel et al., 2013). The expression of EMT-associated genes is a major determinant of melanoma metastasis (Alonso et al., 2007), suggesting that this pathway might be involved in promoting melanoma dissemination. Indeed, RNA-Seq analysis of CTCs isolated from a B-RAF/PTENmutant mouse melanoma model, compared with primary tumor cells, shows the upregulation of EMT signatures coincident with genes implicated in tumor invasiveness (Luo et al., 2014). Similarly, glioblastoma CTCs isolated from patients as well as from a PDX mouse model, when compared with their matched parental tumor, show the upregulation of RNA encoding for the mesenchymal genes SERPINE1, TGFB1, TGFBR2, and vimentin. Overexpression of EMT regulators is observed in the mesenchymal subset of GBMs and is associated with therapeutic resistance and poor clinical outcome (Bhat et al., 2013;Colman et al., 2010;Phillips et al., 2006).

Potential genomic changes induced by EMT
The plasticity of EMT in epithelial cancer cells is generally attributed to epigenetic changes which are reversible (Bedi et al., 2014;McDonald et al., 2011). During development, proliferation is strictly restricted in gastrulating cells undergoing EMT; initiation of proliferation in these cells during EMT leads to severe developmental defects (Murakami et al., 2004;Seher and Leptin, 2000). The incompatibility between EMT and proliferation is also observed in cancer cells. TGF-b and SNAIL-induced EMT in proliferating mammary epithelial cells are associated with mitotic defects leading to binucleate cells and extensive chromosome missegregation (Comaills et al., 2016). These mitotic defects are mediated through TGF-b and SNAIL-mediated suppression of multiple nuclear envelope proteins including nuclear lamins, which maintain the nuclear architecture and orchestrate nuclear transport as well as mitotic processes. The mitotic aberrations induced during EMT are reversible; however, the resulting genomic instability leads to heritable changes. Single-cell RNA-Seq analysis of breast and prostate CTCs shows a correlation between TGF-b and EMT signatures and aneuploidy. Furthermore, binucleated and micronuclei harboring cells were prevalent in CTCs of mesenchymal lineage compared to epithelial lineage, suggesting that EMT might be a mechanism leading to genomic instability. These findings that are consistent with developmental models (Murakami et al., 2004;Seher and Leptin, 2000) might explain the drug resistance associated with EMT (Maheswaran and Haber, 2015;Nieto et al., 2016). In fact, TGF-b activation in the CD44+/CD24À stem-like cells increases DNA copy number alterations and contributes to changes in drug responses (Pal et al., 2017). Taken together, these findings suggest that EMT in proliferating cancer cells could be a major contributor to the molecular evolution and heterogeneity of tumors during disease progression.

Clinical implications of epithelialto-mesenchymal transition in circulating tumor cells
EMT provides a useful model to understand the mechanisms underlying tumor cell invasion and motility, and CTCs offer the possibility of capturing tumor cells during an intermediate step of cancer metastasis; therefore, the overlap of these two concepts will provide a more comprehensive understanding of metastasis. Importantly, though, the study of EMT in CTCs likely has clinical implications that are key to the development of novel cancer treatments and improved deployment of our current therapies. Markers of EMT in CTCs have the potential to provide both prognostic and predictive information. As discussed above, in breast cancer, the number of mesenchymal CTCs has been linked to disease progression and decreased overall survival (Polioudaki et al., 2015;Yu et al., 2013). In colorectal cancer, a study of CTCs in over a thousand patients found that biphenotypic and mesenchymal CTCs correlated with clinical stage, lymph node, and distant metastases (Zhao et al., 2017). Interestingly, a study of metastatic colorectal and prostate cancer patients observed that cell surface vimentin-positive CTCs that expressed nuclear programmed death-ligand 1 (PD-L1) are associated with decreased survival, suggesting that there may exist subpopulations within the mesenchymal CTCs that correlate with clinical parameters (Satelli et al., 2016). In addition to potential prognostic markers of disease, markers of EMT in CTCs also have the potential to provide predictive information regarding a patient's response to therapy. Currently, this area of research is in its early stages. For example, in a recent clinical study of metastatic colorectal cancer, enumeration of CTCs with cell surface vimentin correlated with disease response to postsurgery chemotherapy, suggesting that markers of EMT in CTCs may also prove to be useful markers to predict response to therapy (Satelli et al., 2015b). In a preclinical model of breast cancer metastasis, FOXC2 has been identified as an inducer of EMT and an important regulator of metastasis (Werden et al., 2016). FOXC2 induction of EMT is dependent on MAP kinase activity, and in this study, an inhibitor of the p38 MAP kinase inhibited metastatic spread induced by FOXC2. Therefore, FOXC2 in CTCs may represent a potential marker predicting response to MAP kinase inhibitors. Future studies and investigation of additional markers will be needed.

Future directions
The study of EMT and the parallels between development and cancer have provided significant insight into the metastatic process. The recent advancements in the isolation and characterization of CTCs provide novel tools and information about how EMT contributes to cancer spread. However, one of the challenges of the current studies of both EMT and CTCs is the lack of standardization. For instance, the classification of epithelial, mesenchymal, and biphenotypic cells varies depending on the study even within the same cancer type. Similarly, the isolation of CTCs varies significantly based on the method and the markers used to purify these cells. It will be important to standardize the classifications and methods in order to increase the robustness of our current knowledge. With this in mind, it will be important to reliably establish the degree of heterogeneity of CTCs and clearly define their subpopulations. A consistent isolation and classification method will permit the accurate tracking of changes in CTCs and allow for testing of potential biomarkers of disease progression or response to therapy. Once biomarkers are identified and a reliable method of detection is established, prospective trials testing these biomarkers will be required to establish their clinical usefulness and validity. In addition to the focus on clinical tools based on EMT in CTCs, improved single-cell technologies will continue to advance our understanding of CTCs. For example, it is likely that the expression of mesenchymal genes and misexpression of developmental EMT regulators are accompanied by epigenetic changes that activate these typically repressed genes. Epigenetic regulators are potential novel drug targets that offer a unique mechanism of action that could alter the cellular plasticity. Future investigations will need to further define the role of EMT in CTC generation, survival, extravasation, and colonization of distant organs with the hope of developing new therapies and using our current therapy more effectively.


extfieldsofstudy: Chemistry Medicine

text:
Acetazolamide Suppresses Multi-Drug Resistance-Related Protein 1 and P-Glycoprotein Expression by Inhibiting Aquaporins Expression in a Mesial Temporal Epilepsy Rat Model

Background Mesial temporal epilepsy (MTLE) is the most common type of focal epilepsy in adults, and is often drug-resistant. This study investigated the effects of aquaporins (AQP) inhibitor on multi-drug-resistant protein expression in an MTLE rat model. Material/Methods The MTLE rat model was established by injecting pilocarpine into rats. The MTLE rats were divided into an MTLE-6 h group, an MTLE-12 h group, and an MTLE-24 h group, together with a normal saline group (NS), to examine the AQP4 expression by using Western blot assay and immunohistochemistry assay. The other 18 MTLE model rats were used to observe the effects of the AQP4 inhibitor, acetazolamide, on the multi-drug-resistant protein 1 (MRP1) and P-glycoprotein (Pgp) by using Western blot and immunohistochemistry assays, respectively. Results AQP4 expression was enhanced in hippocampal tissues of MTLE model rats compared to NS rats (P<0.05). More positively stained AQP4 was discovered in hippocampal tissues of MTLE model rats. AQP4 inhibitor significantly decreased multi-drug-resistant protein MRP1 and Pgp expression in the AQP4 inhibitor Interfere group and the AQP4 inhibitor Therapy group compared to the TMLE model group (P<0.05). Conclusions The present findings confirm that the AQP4 inhibitor, acetazolamide, effectively inhibits the multi-drug-resistant protein, MRP1, and Pgp, in the MTLE rat model.

Background
In clinical practice, focal epilepsy is considered as an acquired disease secondary to partial lesions, including trauma, hippocampal sclerosis, tumors, malformations, and vascular malformations of cortical development [1]. According to previous studies [2,3], there are 2 main forms of focal epilepsy: mesial temporal epilepsy (MTLE) and extratemporal epilepsy. MTLE is the most common type of focal epilepsy in adults, and it is drugresistant partial epilepsy in approximately 60% of adults [4]. Extratemporal epilepsy is also a common form of focal epilepsy, and is also treatment-resistant [5]. However, MTLE has become refractory to medical therapy in approximately 60% of patients. Few studies have investigated the pathogenic mechanism of MTLE. Therefore, we performed a preliminary study of MTLE-associated mechanisms.
Previous studies reported that MTLE always results from the damaged or impaired hippocampal network induced by a few initial brain insults [4,6,7]. There are a series of processes mediating the epilepsy-induced changes of hippocampal physiology [8][9][10]. Generally, a prolonged generalized seizure induces neuron injury or neuron death in brain tissues, whereas a generalized seizure damages the hippocampus and impairs memory. Baik et al. [11] reported that neuronal loss and damage in the temporal lobe and hippocampus play important roles in the long-term sequela of recurrent seizures, which has been proved to be related to the progression of MTLE. Kahane and Bartolomei [12] found that seizures derive from a series of multi-structural epileptogenic zones, including the amygdala, entorhinal cortex, and hippocampus, and limbic structures are altered in the temporal lobe.
According to the above descriptions, MTLE mainly damages the hippocampus, and some MTLE is drug-resistant in clinical practice. Therefore, discovering an anti-drug-resistant and hippocampus-protective medicine is critical for MTLE therapy. In recent years, many multi-drug transporters have been discovered, among which the family of multi-drug resistance-related proteins (MPRs) and P-glycoprotein (Pgp) are the most frequently studied [13,14]. The overexpression or up-regulation of the MPRs, including MPR1 and MPR2, have recently been reported in many human refractory epilepsy patients [15]. Tishler et al. [16] found that hippocampal sclerosis-related epilepsy mainly involves the overexpression of MRP1 and Pgp protein in a few regions of the hippocampus.
Aquaporins (AQP) are a series of specific water channels, among which AQP4 is the most important member of the AQP family [17]. AQP4 mainly is located in brain tissues; it plays a critical role in regulating water transportation, and participates in central nervous system (CNS) physiological processes [18][19][20]. Previous studies also showed that the dysregulation of AQP4 expression is correlated with a few forms of epilepsy [21][22][23].
Song et al. [24] proved that the inhibition of AQP4 expression relieves symptoms of epilepsy. The AQP4 plays a critical role in the activation of astrocytes [25], which can also up-regulate the expression of resistance-related proteins MRP1 and P-gp [26]. Therefore, we speculated that AQP4 participates in the occurrence and development of MTLE, which may be of clinical importance for treating multi-drug-resistant MTLE. Many AQP4 inhibitors have recently been discovered, including acetazolamide, sumatriptan, and rizatriptan. Recent studies [27,28] reported that acetazolamide is an effective inhibitor. The AQP4 inhibitors are important factors regulating AQP4 expression. Therefore, we speculated that the investigation of AQP4 inhibitors regulating AQP4 expression may help to develop a novel drug for treatment of MTLE.
We observed the expression of AQP4 in rats undergoing treatment with acetazolamide in brain tissues of an established MTLE rat model. Then, the MTLE model rats were injected with the AQP4 inhibitor to regulate AQP4 expression and to evaluate its effects on the expression of multi-drug-resistance-associated proteins in MTLE model rats.

Animals and MTLE
Thirty-two Sprague-Dawley (SD) rats (6-8 weeks old) weighing 220-250 g were purchased from the Animal Center of Nanjing Medical University. The SD rats were maintained under the conditions of 24-25°C, 50-60% humidity, and light/dark cycle of 12/12 h, with free access to rat chow and water. All animal experiments in this study were approved by the Ethics Committee of Nanjing Medical University, Nanjing, China.

Model establishment and trial grouping
The SD rats were divided into a normal saline group (NS, n=8) and an experimental group (n=24). In the experimental group, the SD rats were pre-treated with the lithium chloride (125 mg/kg, i.p.) to enhance the effects of the pilocarpine. At 18-20 h later, scopolamine (1 mg/kg, i.p.) was injected into the rats to decrease the peripheral effects of the pilocarpine injection. Then, 30 min after the scopolamine injection, pilocarpine was administered i.p. at a dose of 10 mg/kg every 30 min until the occurrence of MTLE. The SD rats were observed and monitored for seizure behaviors at least for 3 h. Seizures were scored and classified according to the Racine score [29]. Criteria for determining the successful establishment of the MTLE model were continuous limbic seizures with scores of 3-5 and lasting for at least 1 h. After 60-90 min of continuous seizures, 10% chloral hydrate (0.3 ml/100 g) was injected to terminate the continuous seizures.

Acetazolamide therapy trial and grouping
To observe the effects of acetazolamide on the MTLE model rats, acetazolamide was applied. Firstly, the MTLE rat model was established as detailed above and the MTLE model was successfully established in 18 rats. The MTLE model rats were divided into 3 groups: the MTLE model group (injected with 35 mg/kg saline, n=6), the acetazolamide interfered group (Interfere group, injected with acetazolamide 35 mg/kg for 7 continuous days, n=6), and the acetazolamide therapy group (Therapy group, injected with acetazolamide 35 mg/kg per day for 28 continuous days, n=6). The normal saline-treated MTLE group (NS group, injected with 35 mg/kg saline, n=6) was defined as the normal control group.

Sample preparation
The MTLE model and NC rats were anesthetized using 10% chloral hydrate (0.3 ml/100 g) and decapitated. The hippocampus tissues were isolated and fixed with 4% paraformaldehyde, and sliced into the DG, CA1, and CA3 regions. Finally, the hippocampus was stored at -80°C. A portion of the fixed hippocampus tissue (n=4 for every group) was cut into a series of 4-μm-thick horizontal sections for the following immunohistochemistry assays.

Immunohistochemistry assay and image analysis
The paraformaldehyde-fixed sections were embedded in paraffin, and sectioned into 4-µm-thick sections. The paraffin was removed by using the regular method [30], and the sections were used for immunohistochemical analysis. The sections were blocked with 5% BSA for 20 min, and incubated with rabbit anti-rat AQP4, GAPDH, MRP1, and P-glycoprotein 1 polyclonal antibody at 4°C overnight. Then, slices were incubated with goat anti-rabbit HRP-labeled IgG at room temperature for 1 h. The immunohistochemistry was conducted using a commercial SP immunohistochemistry kit (KeyGen BioTech., Jiangsu, China). Finally, the sections were immersed in alkaline phosphataselabeled diaminobenzidine (DAB, KeyGen BioTech., Jiangsu, China), and the images were captured using a biological inverted fluorescence microscope (Model: IX51; Olympus, Japan).
The brown granules in the immunohistochemistry images were considered as the AQP4-, MRP1-, and P-glycoprotein 1-positively stained cells. Image Pro Plus 6.0 software (Media Cybernetics, Inc., Bethesda, MD, USA) was used to quantitatively analyze the images. The images were captured at the amplification of 400×, and 5 visual fields were selected randomly for every section. Then, the sections were stained with Ponceau-Victoria blue B, and the integrated optical density (IOD) values of AQP4, MRP1, and P-glycoprotein 1 were evaluated. The mean optical density of 5 visual fields was calculated by the following formula: IOD Sum (total IOD in 5 visual fields)/5 (visual fields).

Carbonic anhydrase levels measurement
We excluded the inhibitive effects of acetazolamide (carbonic anhydrase inhibitor) on the carbonic anhydrase levels in rats. Serum carbonic anhydrase levels were evaluated using a specific ELISA kit (B&D Systems, Minneapolis, MN, USA). In brief, the rabbit anti-carbonic anhydrase antibody was pre-coated onto the micro-titer wells. Aliquots of the serum were added to every well, followed by peroxidase-conjugated antibody to carbonic anhydrase. The color was developed with the hydrogen peroxide and tetramethylbenzidine peroxidase and the absorbance at 450 nm was measured. Wavelength correction was also performed by using the absorbance at 570 nm. Finally, the concentration of carbonic anhydrase was evaluated by interpolation from the standard curve.

Statistical analysis
All data were analyzed with SPSS software 19.0 (SPSS Inc., Chicago, IL, USA). Data are described as mean ±SD of at least 3 independent experiments. The t test was used for statistical comparison between 2 groups, and Tukey's post hoc test was used for multiple-group comparisons. P value less than 0.05 was regarded as a significant difference.

AQP4 expression was enhanced in hippocampal tissues of MTLE model rats
We examined AQP4 expression in DG, CA1, and CA3 regions of the hippocampus by Western blot assay. The results indicated that the AQP4 expression was obviously enhanced in the MTLE model group compared to the NS group at both 12 h and 24 h after the occurrence of seizures ( Figure 1A). According to the results of the different hippocampal regions, AQP4 was significantly enhanced in the CA1 region ( Figure 1B

More positively stained AQP4 was discovered in hippocampal tissues in MTLE model rats
To observe the distribution of APQ4 protein in different regions of the hippocampus, we used immunohistochemistry assay. The results showed that the distribution of positively stained AQP4 protein in the MTLE group was obviously increased compared to the NS group (Figure 2A). Based on the statistical analysis, the positively stained AQP4 was significantly increased in the CA1 region ( Figure 2B), CA3 region ( Figure 2C), and DG ( Figure 2D) of MTLE model rats compared to the NS group (P<0.05).

AQP4 inhibitor decreases multi-drug-resistant protein MRP1
First, we evaluated the inhibitive effects of acetazolamide (carbonic anhydrase inhibitor) on serum carbonic anhydrase   Figure 1). Therefore, we used acetazolamide in the following experiments.
In this study, we injected the AQP4 inhibitor, acetazolamide, into the MTLE model rats. Western blot assay ( Figure 3A) results indicated that MRP1 expression was significantly decreased in the Interfere group (P<0.05) and Therapy group (P<0.01) compared to the NS group ( Figure 3B). The immunohistochemistry assay ( Figure 4A) also showed that the positively stained MRP1 was also obviously decreased in the Interfere group and Therapy group. Statistical analysis showed significantly different levels of positively stained MRP1 in the Interfere group (P<0.01) and Therapy group (P<0.01) compared to the NS group ( Figure 4B).

AQP4 inhibitor inhibits multi-drug-resistant protein Pgp expression
The other multi-drug-resistant protein, Pgp, was also evaluated. The results indicated that Pgp expression was significantly inhibited during treatment with AQP4 inhibitor (acetazolamide, both Interfere group and Therapy group) compared to the NS group, according to Western blot results ( Figure 3C, P<0.01). Moreover, the immunohistochemistry assay also proved that Pgp expression was decreased in the Interfere group (P<0.01) and Therapy group (P<0.01) compared to the NS group ( Figure 4C).

Discussion
Many studies [31][32][33] have confirmed that multi-drug-resistant proteins are extensively over-expressed during epileptogenesis, both in the MTLE model and in epilepsy patients, especially in hippocampal brain tissues. Therefore, the inhibition of multi-drug-resistant proteins in the MTLE model or epilepsy patients may enhance the therapeutic effect of anti-epilepsy drugs, which is a critical strategy for the development of drugs for treatment of epilepsy.  AQP4 is mainly expressed in the hippocampal brain tissues, which includes M1 and M23 subtypes [17,34]. AQP4 mediates transportation of water in brain tissues, which maintains physiological functions of the CNS. A previous study [35] reported that AQP4 expression is increased during the process of epilepsy, and is considered as an inducer of cytotoxic cerebral edema in epilepsy. Therefore, we believe that the AQP4 protein plays a protective function in preserving homeostasis in the CNS. The epileptic rat model induced by lithium chloride combined with pilocarpine could be considered as the pathological model to investigate the effects of AQP4 inhibitor on AQP4 expression and regulation of multi-drug-resistance proteins. In our study, AQP4 levels were significantly increased in the MTLE rat model compared to the normal rats, which is consistent with previous studies reported [36,37]. Therefore, epilepsy may be associated with the AQP4-mediated water transportation in the MTLE model. Previous studies [38,39] proved that deficiency or down-regulation of AQP4 expression relieves cerebral edema and improves conditions of local cerebral ischemia caused by toxic water molecule accumulation. Therefore, we applied the AQP4 inhibitor to the MTLE models to suppress the expression of AQP4, and observed the related mechanism for the protective role of AQP4 inhibitor in the MTLE rat model.
In this study, we injected the AQP4 inhibitor, acetazolamide, into the MTLE model rats, which has been used as the antagonist to AQP4 in a few reports [28,40,41]. Our results indicate that acetazolamide significantly inhibits the multi-drug-resistant protein, MRP1, which was first reported in the MTLE rat model. Sun et al. [42] reported that the inhibition of MRP1 enhanced the efficacy of antiepileptic drugs (AEDs) through enhancing local drug availability. Chen et al. [43] also found that the up-regulation of MRP1 protein plays an important role in drug resistance of brain cells to AEDs in the amygdale of kindling rats. Moreover, another multi-drug-resistant protein, Pgp [44,45], has also been evaluated in the MTLE rat model. These findings show that acetazolamide significantly suppresses Pgp expression in the MTLE rat model.   the AQP4-specific inhibitor would be better compared to acetazolamide. Secondly, the present study does not clarify the effects of acetazolamide on the pathology of MTLE and disease progression, and we did not observe the therapeutic effects of acetazolamide.

Conclusions
The AQP4 inhibitor, acetazolamide, could effectively inhibit the multi-drug-resistant proteins, MRP1 and Pgp, in the MTLE rat model. However, the specific mechanism for the AQP4 inhibitor triggered MRP1 and Pgp down-regulation has not been clarified, which would be explored in the future study.

Conflict of interests
None.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Engineering

text:
Biomimetically Engineered Demi‐Bacteria Potentiate Vaccination against Cancer

Abstract Failure in enhancing antigen immunogenicity has limited the development of cancer vaccine. Inspired by effective immune responses toward microorganisms, demi‐bacteria (DB) from Bacillus are engineered as carriers for cancer vaccines. The explored hydrothermal treatment enables the Bacillus to preserve optimal pathogen morphology with intrinsic mannose receptor agonist. Meanwhile, the treated Bacillus can be further endowed with ideal hollow/porous structure for efficient accommodation of antigen and adjuvant, such as CpG. Therefore, this optimal engineered nanoarchitecture allows multiple immunostimulatory elements integrate in a pattern closely resembling that of bacterial pathogens. Such pathogen mimicry greatly enhances antigen uptake and cross‐presentation, resulting in stronger immune activation suitable for cancer vaccines. Indeed, DB‐based biomimetic vaccination in mice induces synergistic cellular and humoral immune responses, achieving potent therapeutic and preventive effects against cancer. Application of microorganism‐sourced materials thus presents new opportunities for potent cancer therapy.

Experimental Section
Characterization of Surface Properties.
Zeta potential of the 4 kinds of hydrothermal treated bacteria was determined by ZetaSizer (Nanoseries, Malvern) in pure water and the surface contact angle was measured by using contact angle meter (OCA-20, DataPhysics).

Evaluation of Loading Efficiency and Release behaviour for OVA and CpG.
For the encapsulation studies, CpG or OVA at certain concentrations was mixed with 50 μg DB in 100 μL PBS at 4 o C for 12 h after vacuum negative pressure treated. Free CpG or OVA was washed away by three centrifugation-redispersion cycles, with all supernatants reserved. The loading amount of CpG or OVA was determined by mass balance between the initial incubation solution and the reserved supernatant. For in vitro release studies, OVA or CpG-loaded DB was incubated in 1 mL PBS (pH 7.4) under agitation at 37 °C. Supernatants were periodically collected by centrifugation and replaced with fresh buffer of equal volume.
The CpG concentration was determined using an Infinite M200 microplate spectrophotometer and a NanoQuant Plate (Tecan), and the OVA concentration was determined by a BCA protein determination kit (Thermo).

Culture of DCs.
DCs were generated by flushing tibia and femurs of 6-8 weeks old male C57BL/6 mice.

Observation of Capture of DB at the DC Membrane.
For SEM imaging, DCs were adhered to poly-L-lysine-coated glass slides, and 2.5 μg mL -1 of DB was then added for 1 h of incubation. The cells were washed thoroughly immediately afterward to remove free DB. Next, the samples were fixed in 2.5% (v/v) glutaraldehyde, dehydrated with gradient methyl cyanide solutions, and gold-sprayed for imaging under a field emission SEM (JEOL JSM6700F).

Intracellular Trafficking of DB.
DCs were incubated with DB for 1 h at 37 o C and washed to remove free DB, followed by further incubation for another 6 h to ensure the synchronisation of intracellular processes.
For early endosome staining, the cells were fixed in 4% (v/v) formaldehyde and permeabilized via a 10 min incubation with 0.2% (v/v) Triton X-100 at room temperature.
Subsequently, 5% (v/v) goat serum was added and incubated for 30 min to block nonspecific adsorption. Staining with a polyclonal rabbit anti-murine EEA1 antibody was performed at 4 °C for 24 h, followed by 30 min of incubation with Texas Red-goat anti-rabbit IgG at room temperature. For lysosome staining, DCs were directly stained with LysoTracker Red for 30 min at 37 o C, without cell fixation and permeabilization. The colocalization ratios (CRs) of DB with early endosomes or liposomes were calculated using Leica LAS Colocalization software.

Preparation and Characterization of Traditional Particulate Adjuvants.
Liposomes were prepared by thin-film hydration method. [1] Briefly, a mixture of 1palmitoyl-2-oleoylphosphatidylcholine (POPC) and cholesterol (in molecular ratio 1:2) were dissolved in chloroform, and a thin-film was formed on the inner side of the round bottom flask by evaporating the solvent under vacuum using a rotavapor. The film was then hydrated using 10 mL of PBS containing 50 ng mL -1 water-soluble Cy5. The formed liposomes were then sonicated using a probe sonicator for 2 min, and then sized by extrusion through a 0.4 μm pore-sized polycarbonate membranes repeatedly. The un-encapsulated Cy5 was then removed by dialysis. PLA NPs and Chitosan NPs were prepared using the Shirasu Porous Glass (SPG) membrane emulsification technique. [2] By selecting a SPG membrane with a certain pore size, the size of the emulsion droplets and subsequent NPs can be well controlled around 500 nm.
To prepare the fluorescent NPs, oil-soluble Cy5 was added together with PLA to the oil phase before the emulsification, free Cy5 was then removed by utterly washed with water. The zeta potential and size distribution were determined by Zetasizer (Nanoseries, Malvrn).

Evaluation of DC Activation and Antigen Presentation.
In total, 1×10 6 cell mL -1 DCs were incubated with different formulations for 24 h in 24well plates. At the end of the incubation, the DCs were collected by centrifugation (500 g, 3 min) and stained with antibody for 30 min at 4 o C. The following fluorophore-conjugated antibody reagents (purchased from BioLegend and eBioscience) were used: APC-Cy7-CD11c, PE-CD40, FITC-CD80, PE-CD86, PerCP-eFluor 450-MHC II, and APC-SIINFEKL-MHC I.
Subsequently, the cells were washed with PBS, and the levels of surface costimulatory molecules and recognition signals for T cell activation were analysed by FC. Otherwise, to monitor the cytokine profile of the DCs, cell culture supernatants were collected, and the secretion levels of IL-6, IL-12, TNF-α, MCP-1, and IFN- were detected using a CBA Mouse Inflammation Kit (BD) to quantify. [3] Briefly, capture beads and phycoerythrin (PE) detection reagent were incubated with standard samples or test samples for 2 hours, washed in wash buffer. After acquisition of sample data using the flow cytometer, the sample results were tabulated and graphed using the BD CBA Analysis Software.

Analysis of PRR mRNA Expression.
RNA was isolated with TotalRNAExtractor (Sangon Biotech, Shanghai, China) from naive DCs and DC treated by DB, CpG, or DB:CpG for 24 h. RNA samples were then DNase I digested, reverse transcribed with iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, Canada), and analysed by qPCR with Ssofast EVI Green Supermix (Bio-Rad Laboratories, Hercules, Canada). Data was displayed as relative expression compared to GAPDH. PCR products were sequence verified. The sequences of primers for target genes and internal control gene GAPDH are listed in the table below.

In-vivo Fate of Antigen.
To by Vectra platform and the recruited cells were quantitated by using the inform software (Caliper Life Sciences, Hopkinton, USA). [4] Analysis of DC Migration to Lymph Nodes.
To track in vivo DC migration from injection site towards lymph nodes, the draining lymph nodes (LNs) were harvested at 24 h after immunisation. Cell suspensions from LNs were prepared by mechanical disruption and pressing of the tissue through 70 μm cell strainers. The CD11c + SIINFEKL-MHC I + cell numbers among CD11c + cells (DCs) were examined after staining by APC-Cy7-CD11c and APC-SIINFEKL-MHC I mAbs.

Preparation of Splenocytes and CD8 T Cells.
Spleen of 6-8 weeks old male C57BL/6 or OT-1 mice was harvested and ground to prepare single-cell suspension. After red blood cell lysis, cells were washed and cultured in complete RPMI 1640 medium containing 10% (v/v) heat-inactivated FBS. For CD8 T cell enriching, splenocytes were sorted by Dynabeads (Life technologies) to deplete CD8-negative leucocytes, and CD8T with purity around 90% could be harvested.

Evaluation of Health Condition.
To examine the T cell-subset distribution after the treatment of developed tumors, mice

Quantitative Determination of CTL and Treg Cell in Tumor.
To  For all groups, the initial particle number in culture medium was normalized to that of DB (2.5 μg mL -1 ).
(c) Comparative internalization profiles of free, DB-mixed, and DB-loaded CpG and OVA. DC uptake of both guest agents was found to be greatly improved by encapsulation in DB, enhanced intracellular delivery for both CpG and OVA could be achieved simultaneously.
(d) Effect of the MRs on the uptake of DB by DCs. Relative DC uptake of DB prepared via different hydrothermal treatment times was shown in left, to determine the proportion of MRmediated endocytosis, mannose (200 μg mL -1 ) was added to block the MRs 1 h before coincubation. The data of MR-blocked group in were normalized to the internalization in the corresponding control group (left). These results confirmed that the MR ligand on 6hDB was sufficient to significantly promote the interaction with APCs. Data in all groups represent the mean ± s.d. of three independent experiments with n=3. *p<0.05, **p<0.01.    Mice with established tumors were treated with different formulations, blood samples were collected before scarification on day 23 (for developed tumor therapy), and the serum AST, ALT, BUN, LDH, and ALP levels were determined. The data are represented as the mean ± s.d. with n=7. Among these parameters, AST and ALT are specific indicators of hepatic toxicity, and LDH is a parameter related to injury of major organs, including the liver, heart, and kidney. In contrast with the abnormally enhanced levels in the other groups, the serum AST and ALT concentrations in the 2×DB:CpG/OVA group returned to normal ranges. A similar result was observed for the LDH levels, which indicated that treatment with the biomimetic vaccine had effectively protected the mice from hepatic or other organ damage. Together, all these data showed that mice that received the biomimetic vaccine were in much better health and had a better prognosis.


text:
Elevated Cardiovascular Risks among Postmenopausal Women: A Community Based Case Control Study from Nepal

Cardiovascular disease (CVD) is one of the leading causes of death worldwide which is more prevalent in women after menopause. Hormonal changes associated with menopause are accountable for dyslipidemic pattern that causes CVD and associated complications. Therefore, the present study was commenced to compare the lipid profile in pre- and postmenopausal women. A descriptive cross-sectional study was conducted at Manmohan Memorial Institute of Health Sciences (MMIHS) from February 2016 to July 2016. A total of 260 fasting samples were collected from healthy women, 130 from premenopausal and 130 from postmenopausal women, and analyzed for Total Cholesterol (TC), Triacylglycerol (TAG), High Density Lipoprotein Cholesterol (HDL-C), and Low Density Lipoprotein Cholesterol (LDL-C) as per the guideline provided by the reagent manufacturer (Human, Germany). All the parameters were analyzed by Stat Fax 3300 semi auto analyzer. TC, TAG, HDL-C, and LDL-C were highly significantly increased in postmenopausal women when compared to premenopausal women. LDL/HDL ratio was significantly elevated in postmenopausal women than in premenopausal women. BMI was significantly positively correlated with TC and TAG in both pre- and postmenopausal population and it was positively correlated with HDL-C in premenopausal population while negatively correlated in postmenopausal population. Since more of the atherogenic lipid parameters are increased in postmenopausal women, they appear to be more prone to have CVD and associated complications in near future. Hence, it is mandatory to monitor and manage dyslipidemic pattern in every woman experiencing menopause.

Background
Cardioprotective effect on premenopausal women is believed to be imposed by adequacy of endogenous estrogen level produced during menstrual cycle. This could be the possible reason for declined rate of coronary heart disease in fertile women than men [1]. However, by the end of reproductive life ovaries fail to produce significant amount of estrogen instigating postmenopausal women more prone to disease associated with estrogen deficiency like heart diseases, osteoporosis, and dyslipidemia [2,3]. Hormonal changes after menopause such as low plasma estrogen level and elevated Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) level have significant effect on plasma lipid and lipoprotein metabolism resulting in ultimate cardiac related disorders [4,5].
Estrogen shows cardioprotective effect by maintaining high level of HDL-C and low level of LDL-C and TAG. Mass clearance of LDL-C from the plasma results probably from accelerated conversion of hepatic cholesterol to bile acids and increased expression of LDL receptors on cell surfaces. Increase in production of apolipoprotein A-I and decrease in hepatic lipase activity facilitate increase of HDL-C [6]. Estrogen causes hepatic expression of apoprotein gene; consequently it affects lipid and lipoprotein metabolism [1].
Such cardio defensive effect is lost after menopause driving postmenopausal women towards high risk of debilitating and often fatal complications of cardiovascular disease (CVD) [7]. Estrogen hormone regulates cellular level directly by mRNA production for specific protein including proteins for lipid metabolism such as lipoprotein lipase (LPL) and hormone sensitive lipase (HSL) in adipose tissue. Estrogen also has indirect action on adipose tissue by stimulating release of other hormones such as growth hormone (GH), catecholamine, and glucagon which increase activity of HSL. 17beta-estradiol (a major circulating form of estrogen) regulates the rate of synthesis of structural apolipoproteins for VLDL and HDL in liver. It reduces the rate of apoB-100 synthesis, thereby reducing the VLDL concentration which is risk factor for atherosclerosis. Conversely, it enhances the rate of apoA-I and apoA-II synthesis, thereby increasing HDL concentration which is atheroprotective. HDL containing apoA-I and apoA-II helps to degrade cholesterol from VLDL and chylomicron by reverse cholesterol transport from peripheral tissue to liver [8].
TC and TAG contribute major circulating lipid in our body, while chylomicron, VLDL, IDL, LDL, and HDL are the lipoproteins functioning as vehicle for cholesterol transport. Cholesterol content in VLDL, IDL, LDL, and HDL determines the total plasma cholesterol. LDL conveys endogenous cholesterol from the liver to the peripheral tissues. Under certain circumstances, LDL deposit cholesterol in the intimal layer of the arteries, thereby initiating the atherosclerotic process. HDL functions as atheroprotective since it has reverse cholesterol transport activity, thereby removing excess cholesterol from cells, including cholesterol engulfed macrophages in atherosclerotic lesions, and transporting it into the liver for hepatic excretion through bile acid. Onset, progression, and complications of atherosclerotic plaque are determined by the balance between these two lipoproteins [9].
A fatty streak lesion is produced with an accumulation of cholesterol esters in the intimal layer of the arteries, indicating the initiation of atherosclerosis. The fatty streak may progress to atherosclerotic plaque if the process continues over time. If the plaque continues to grow it may block the vessel or form thrombosis and lead to further complication of ischemic disease like ischemic stroke, Coronary Heart Disease, or Peripheral Artery Disease [9].
An estimated 17.5 million people died from CVD in 2012, representing 31% of all global deaths. Over three quarters of CVD death occur in low and middle income countries [10]. According to American Heart Association report (2002), after menopause 70% of women develop cardiovascular disease and 30% develop osteoporosis in USA [3].
Dyslipidemia, especially hypercholesterolemia, is the major risk factor of CVD. Cardiovascular disease is the most vulnerable health problem prevailing in every corner of world including Nepal. In Nepal, women cover beyond 50% of population among which more of the population are over the age of 50 years. Specially, this group of population lacks physical activities due to their retired life; in addition, consumption of unhealthy food makes them more prone to suffer from atherogenic effects. This is the mean age for menopause suggesting that more of the Nepalese women are probably at risk of developing complications associated with cardiovascular disease which is the matter of concern [11].
Since estrogen plays decisive role in lipid and lipoprotein metabolism, it is indispensable to monitor lipid profile in postmenopausal women who tend to have diminished estrogen level. Hence, the present study was undertaken to compare serum level of TC, TAG, HDL-C, and LDL-C in preand postmenopausal women

Methods
A descriptive cross-sectional study was conducted during the period of six months among the female population of Dholahity community, Lalitpur, Nepal. A total of 260 overnight fasting blood specimens (5 ml) were collected from healthy women, 130 from pre-and 130 from postmenopausal women. Serum samples were separated for the analysis of Total Cholesterol (TC), Triacylglycerol (TAG), High Density Lipoprotein Cholesterol (HDL-C), and Low Density Lipoprotein Cholesterol (LDL-C) as per the guideline provided by the reagent manufacturer (Human GmBh, Germany). All the parameters were analyzed by Stat Fax 3300 semi auto analyzer in the Department of Laboratory Medicine, Manmohan Memorial Institute of Health Sciences.

Inclusion and Exclusion Criteria.
Apparently healthy women aged between 22 and 85 years were included in the study. Women who undergone menopause due to hysterectomy or cessation of periods other than by a natural cause, women on hormone replacement therapy (HRT), women having irregular menses, thyroid patients, pregnant and lactating women, diabetic women, heavy smokers, and alcoholic women were excluded.

Ethical Approval. Written permission was taken from
Institutional Review Committee (IRC) of Manmohan Memorial Institute of Health Sciences (MMIHS) and informed consent was taken from each individual.

Data Analysis.
Data were analyzed using SPSS version 20.0 and Microsoft Excel 2013. Student's -test was used to analyze differences in lipid profile between pre-and postmenopausal groups. Pearson correlation coefficient was used to evaluate the association of BMI with lipid parameters.

Results
The study was carried out in 260 healthy women; among them 130 were premenopausal and 130 were postmenopausal. The average age of premenopausal group was 35.96 ± 8.6 and that of postmenopausal group was 64.5 ± 11.1. Serum TC, TAG, HDL-C, and LDL-C levels were found to be statistically significantly increased in postmenopausal women compared to premenopausal women ( value < 0.05) ( Table 1).
The atherogenic indices TC/HDL and TAG/HDL were found to be increased but not significantly in postmenopausal women compared to premenopausal women. However, LDL/HDL ratio was found to be statistically significantly increased in postmenopausal women than in premenopausal women. We found that 18.4% of postmenopausal women were under cardiovascular risk as defined by TC/HDL indices greater than 5 ( Table 2).
BMI was significantly positively correlated with TC and TAG in both pre-and postmenopausal women. Besides,   BMI was found to be negatively correlated with HDL-C in postmenopausal women while positively correlated in premenopausal women (Table 3).

Discussion
Low and middle income countries are in the peril of noncommunicable disease which is almost half of the total disease burden. Cardiovascular disease solely attributed for 30% of death worldwide in 2005 only [12]. Coronary Artery Disease (CAD) among men is more prevalent than women up to the age of 50 years, but then the incidence is overturned afterwards causing increased prevalence of CAD in women [13]. Most probable reason behind this could be the loss of cardioprotective effect of estrogen which is slowly downturned in women after menopause. Therefore, it necessitates screening of every women undergoing menopause for early diagnosis and prevention of CVD especially in the economically poor countries like Nepal where more than half of the population are women.
This time framing descriptive cross-sectional study puts emphasis on comparison of the pattern of lipid profile in preand postmenopausal women. The study also highlights the atherogenic indices on the two groups.
Out of total 260 healthy women, 130 were premenopausal women aged from 22 to 50 years and 130 were postmenopausal women aged from 45 to 85 years. Serum TC, TAG, HDL-C, and LDL-C levels were found to be statistically significantly increased in postmenopausal women in comparison with premenopausal women ( value < 0.05) in our study. Almost all study shows increased levels of TC, TAG, and LDL-C in postmenopausal group compared to premenopausal group but there is a wide controversy regarding changes in HDL-C after menopause. Our result was quite similar to the study conducted by Ifueko (2013), in which TC, TAG, HDL-C, and LDL-C were increased significantly with " " value < 0.001 in postmenopausal women than in premenopausal women [14]. Likewise, Shenoy and Vernekar (2015) also found significantly increased TC, TAG, and LDL-C but the HDL-C was not increased significantly in postmenopausal women in comparison to premenopausal women [15]. Similarly, a study done by Derby et al. (2009) showed a result of significant increase in TC and LDL-C while TAG was increased nonsignificantly and there was no changes in HDL-C in postmenopausal women when compared to premenopausal women [16]. A study done by Peters et al. in 1999 found significantly increased TC and LDL-C but the increase in TAG and HDL-C was nonsignificant in postmenopausal women than in premenopausal women [17]. Another similar study done by Berg et al. (2004) found nonsignificant increase in TC and TAG, no change in HDL-C, and significant increase in LDL-C in postmenopausal women when compared to premenopausal women [13]. Likewise, Igweh et al. in 2005 showed nonsignificant increase in TC and TAG, significant decrease in HDL-C, and significant increase in LDL-C in postmenopausal women when compared to premenopausal women [18]. Also a study done by Sapkota et al. in 2015 in Nepal showed significantly increased TC, TAG, and LDL-C which is in agreement with the present study and significantly decreased HDL-C in postmenopausal women than in premenopausal women which is in contrast to the present study [11].
A longitudinal study done by Derby et al. showed increasing trend of TC from premenopausal through menopausal transition to postmenopausal status [16]. Though various factors influencing cholesterol are prevalent, most detrimental cause of increase in TC level in postmenopausal women could be alteration in the cardioprotective sex hormone especially estrogen and progesterone. Also some studies have revealed the occurrence of great genetic variance for most lipids [3].
Though the level of TAG is increased in postmenopausal women few studies found that the increase was not significant. This variation in increasing pattern may be due to variation in study population and different age group of postmenopausal women recruited in the study. The possible reason behind the increase in TAG in postmenopausal women is the effect of estrogen on adipocyte cells and lipoprotein lipase activity. Estrogen deficiency will enhance the increase in adipocyte cell size and also increases the activity of lipoprotein lipase [3].
There is vast controversy in the level of HDL in postmenopausal women. Some studies revealed the significant decrease in level of HDL in postmenopausal women while some studies revealed significant increase in level of HDL in postmenopausal women supporting the present study. This discrepancy result may be due to variation in study population, life intervention, and duration of menopause. A longitudinal study done by Derby et al. showed there was gradual increase in HDL from premenopause through menopausal transition to postmenopause. However, there was slight decrease in HDL level at late postmenopause [16].
Most studies showed significant increase in LDL cholesterol level supporting the present study, without any contradiction. This obvious result observed in all studies may represent the fact that estrogen has direct effect on LDL metabolism. The main reason for the absolute increase in LDL may be due to the decrease in estrogen that stimulated the synthesis of LDL receptor which directly causes reduction of LDL receptor after menopause [3].
In our study, TC, TAG, and LDL-C were positively correlated with BMI while HDL-C was negatively correlated with BMI in postmenopausal women, which may indicate poor dietary and physical activity intervention among our postmenopausal population.
The atherogenic indices were obtained in the present study which showed TC/HDL with average of 3.5 in premenopause and 3.7 in postmenopause while another similar study done by Ifueko showed a contrast result with 2.68 and 2.39 in pre-and postmenopause, respectively. This shows that the present study population group is more at risk of having complications associated with cardiovascular disease. Furthermore, the LDL/HDL ratio in present study was found to be average of 1.9 and 2.2 in pre-and postmenopause, respectively, while the other similar study done by Ifueko found result of 0.95 and 0.84 in pre-and postmenopause, respectively, which was dissimilar to the present finding.
Though both HDL-C and LDL-C were found to be significantly increased in postmenopausal women as compared to premenopausal, significantly increased LDL/HDL ratio in postmenopausal women implied that atherogenic cholesterol, LDL-C, is aggregating more rapidly than good cholesterol, HDL-C. This indicates that the postmenopausal women in study population are more prone to cardiovascular risk.

Conclusion
Potentially, adverse changes in lipid profile along with significant increase in cardiac risk ratio in postmenopausal women of the study remark that this group of women is at increased risk of having complications associated with cardiovascular disease in near future. Early and timely detection and primary prevention can avoid morbidity and mortality in this high risk population. Moreover, positive correlation of BMI with TC and TAG in pre-and postmenopausal women concludes that dietary interventions and physical activity are to be encouraged in both the study groups especially when other cardiac risk factors are prevailing.