Validation of reference genes for normalization of real-time quantitative PCR studies of gene expression in brain capillary endothelial cells cultured in vitro.

BACKGROUND: The genes encoding ß-actin and GAPDH are two of the most commonly used reference genes for normalization in in vitro blood-brain barrier studies. Studies have, however, shown that these reference genes might not always be the best choice. The aim of the present study was to evaluate 10 reference genes for use in mRNA profiling studies in primary cultures of brain endothelial cells of bovine origin. METHODS: Gene evaluations were performed by qPCR in mono-culture and in co-cultures with astrocytes. The expression of reference genes was furthermore investigated during culture. Qbase+ software was used to analyze the stability of the tested genes and for determinations of the optimal number of reference genes. RESULTS: The stability of the reference genes varied between the culture configurations, but for all culture configurations we found that the optimal number of reference genes were two. PMM-1, RPL13A and ß-actin were the most stable genes in mono-cultures, non-contact co-culture and contact co-culture respectively. For studies comparing gene expression between different culture configurations the optimal number of reference genes was three and RPL13A was found to be most stable. During cell culture a number of four reference genes were found to be optimal and YWHAZ was found to be the most stable gene. ß-actin and GAPDH were found to be the least stable genes during culture. CONCLUSION: Overall we found that the validation of reference genes was important in order to normalize target gene expression correctly, and suggest sets of reference genes to be used under different experimental conditions, in order to quantify mRNA transcript levels in blood-brain barrier cell models correctly.

The insulin receptor is expressed and functional in cultured blood-brain barrier endothelial cells but does not mediate insulin entry from blood to brain.

Insulin and its receptor are known to be present and functional in the brain. Insulin cerebrospinal fluid concentrations have been shown to correlate with plasma levels of insulin in a nonlinear fashion, indicative of a saturable transport pathway from the blood to the brain interstitial fluid. The aim of the present study was to investigate whether insulin was transported across brain endothelial cells in vitro via an insulin receptor-dependent pathway. The study showed that the insulin receptor was expressed at both the mRNA and protein levels in bovine brain endothelial cells. Luminally applied radiolabeled insulin showed insulin receptor-mediated binding to the endothelial cells. This caused a dose-dependent increase in Akt-phosphorylation, which was inhibited by coapplication of an insulin receptor inhibitor, s961, demonstrating activation of insulin receptor signaling pathways. Transport of insulin across the blood-brain barrier in vitro was low and comparable to that of a similarly sized paracellular marker. Furthermore, insulin transport was not inhibited by coapplication of an excess of unlabeled insulin or an insulin receptor inhibitor. The insulin transport and uptake studies were repeated in mouse brain endothelial cells demonstrating similar results. Although it cannot be ruled out that culture-induced changes in the cell model could have impaired a potential insulin transport mechanism, these in vitro data indicate that peripheral insulin must reach the brain parenchyma through alternative pathways rather than crossing the blood-brain barrier via receptor mediated transcytosis.

Clinical and Regulatory Aspects of Companion Diagnostic Development in Oncology.

Nearly 20 years have passed since the US Food and Drug Administration (FDA) approved the first companion diagnostic and today this type of assay governs the use of 21 different anticancer drugs. The regulators deem these assays essential for the safe and effective use of a corresponding therapeutic product. The companion diagnostic assays are important both during the drug development process as well as essential treatment decision tools after the approval of the drugs.

Companion and Complementary Diagnostics-Focus on PD-L1 Expression Assays for PD-1/PD-L1 Checkpoint Inhibitors in Non-Small Cell Lung Cancer.

Over the last couple of decades, molecular diagnostics have played an increasing role in drug development. Especially within oncology, more and more drugs are being developed together with a predictive biomarker assay using the drug-diagnostic codevelopment model. Not only do these assays support the development process but also the use of the drugs after regulatory approval as an important treatment decision tool. When these predictive biomarker assays are linked to a specific drug, they are called companion diagnostics. Furthermore, these assays are also considered an important element in the realization of precision medicine. Today, 21 different drugs have obtained US FDA approval together with a companion diagnostic assay, and the requirement for testing is part of their regulatory labeling. More than half of these drugs are for treatment of non-small cell lung cancer (NSCLC). With the approval of the different programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) immune checkpoint inhibitors, for the treatment of advanced stage NSCLC, a new class of predictive biomarker assays-complementary diagnostics-has emerged. Until now, 3 immune checkpoint inhibitors have obtained regulatory approval for treatment of NSCLC, and they all have a biomarker assay linked to their use. However, only for pembrolizumab, the PD-L1 immunohistochemical (IHC) 22C3 pharmDx assay has status as a companion diagnostic. For nivolumab and atezolizumab, the assays PD-L1 IHC 22C3 pharmDx and Ventana PD-L1 (SP142) have status as complementary diagnostics, which means that there are no requirements for testing included in the labeling for these drugs. Here, the authors discuss the clinical performance of the different IHC PD-L1 expression assays including the selection of the clinical cutoff values.

Bidirectional apical-basal traffic of the cation-independent mannose-6-phosphate receptor in brain endothelial cells.

Brain capillary endothelium mediates the exchange of nutrients between blood and brain parenchyma. This barrier function of the brain capillaries also limits passage of pharmaceuticals from blood to brain, which hinders treatment of several neurological disorders. Receptor-mediated transport has been suggested as a potential pharmaceutical delivery route across the brain endothelium, e.g. reports have shown that the transferrin receptor (TfR) facilitates transcytosis of TfR antibodies, but it is not known whether this recycling receptor itself traffics from apical to basal membrane in the process. Here, we elucidate the endosomal trafficking of the retrograde transported cation-independent mannose-6-phosphate receptor (MPR300) in primary cultures of brain endothelial cells (BECs) of porcine and bovine origin. Receptor expression and localisation of MPR300 in the endo-lysosomal system and trafficking of internalised receptor are analysed. We also demonstrate that MPR300 can undergo bidirectional apical-basal trafficking in primary BECs in co-culture with astrocytes. This is, to our knowledge, the first detailed study of retrograde transported receptor trafficking in BECs, and the study demonstrates that MPR300 can be transported from the luminal to abluminal membrane and reverse. Such trafficking of MPR300 suggests that retrograde transported receptors in general may provide a mechanism for transport of pharmaceuticals into the brain.

Transferrin receptor expression and role in transendothelial transport of transferrin in cultured brain endothelial monolayers.

Receptor-mediated transcytosis of the transferrin receptor has been suggested as a potential transport system to deliver therapeutic molecules into the brain. Recent studies have however shown that therapeutic antibodies, which have been reported to cross the brain endothelium, reach greater brain exposure when the affinity of the antibodies to the transferrin receptor is lowered. The lower affinity of the antibodies to the transferrin receptor facilitates the dissociation from the receptor within the endosomal compartments, which may indicate that the receptor itself does not necessarily move across the endothelial cells by transcytosis. The aim of the present study was to investigate transferrin receptor expression and role in transendothelial transferrin transport in cultured bovine brain endothelial cell monolayers. Transferrin receptor mRNA and protein levels were investigated in endothelial mono-cultures and co-cultures with astrocytes, as well as in freshly isolated brain capillaries using qPCR, immunocytochemistry and Western blotting. Transendothelial transport and luminal association of holo-transferrin was investigated using [125I]holo-transferrin or [59Fe]-transferrin. Transferrin receptor mRNA expression in all cell culture configurations was lower than in freshly isolated capillaries, but the expression slightly increased during six days of culture. The mRNA expression levels were similar in mono-cultures and co-cultures. Immunostaining demonstrated comparable transferrin receptor localization patterns in mono-cultures and co-cultures. The endothelial cells demonstrated an up-regulation of transferrin receptor mRNA after treatment with the iron chelator deferoxamine. The association of [125I]holo-transferrin and [59Fe]-transferrin to the endothelial cells was inhibited by an excess of unlabeled holo-transferrin, indicating receptor mediated association. However, over time the cell associated [59Fe]-label exceeded that of [125I]holo-transferrin, which could indicate release of iron in the endothelial cells and receptor recycling. Luminal-to-abluminal transport of [125I]holo-transferrin across endothelial cell monolayers was low and not inhibited by unlabeled holo-transferrin. This indicated that transendothelial transferrin transport was independent of transferrin receptor-mediated transcytosis.

Companion diagnostics-a tool to improve pharmacotherapy.

The variability of pharmacotherapy can be of a significant magnitude, and the main reason for this is often diseases heterogeneity. Patients who have similar diagnoses very often respond differently to the same pharmacological intervention, with great variability in both efficacy and safety outcome. Despite having discussed personalized medicine for more than a decade, we still see that most drug prescriptions for severe chronic diseases are largely based on 'trial and error' and not on solid biomarker data. However, with the advance of molecular diagnostics and a subsequent increased understanding of disease mechanisms, things are slowly changing. Within the last few years, we have seen an increasing number of predictive biomarker assays being developed to guide the use of targeted cancer drugs. This type of assay is called companion diagnostics and is developed in parallel to the drug using the drug-diagnostic co-development model. The development of companion diagnostics is a relatively new discipline and in this review, different aspects will be discussed including clinical and regulatory issues. Furthermore, examples of drugs, such as the ALK and PD-1/PD-L1 inhibitors, that have been approved recently together with a companion or complimentary diagnostic will be given.

Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates.

Using therapeutic antibodies that need to cross the blood-brain barrier (BBB) to treat neurological disease is a difficult challenge. We have shown that bispecific antibodies with optimized binding to the transferrin receptor (TfR) that target ß-secretase (BACE1) can cross the BBB and reduce brain amyloid-ß (Aß) in mice. Can TfR enhance antibody uptake in the primate brain? We describe two humanized TfR/BACE1 bispecific antibody variants. Using a human TfR knock-in mouse, we observed that anti-TfR/BACE1 antibodies could cross the BBB and reduce brain Aß in a TfR affinity-dependent fashion. Intravenous dosing of monkeys with anti-TfR/BACE1 antibodies also reduced Aß both in cerebral spinal fluid and in brain tissue, and the degree of reduction correlated with the brain concentration of anti-TfR/BACE1 antibody. These results demonstrate that the TfR bispecific antibody platform can robustly and safely deliver therapeutic antibody across the BBB in the primate brain.

An electrically tight in vitro blood-brain barrier model displays net brain-to-blood efflux of substrates for the ABC transporters, P-gp, Bcrp and Mrp-1.

Efflux transporters of the ATP-binding cassette superfamily including breast cancer resistance protein (Bcrp/Abcg2), P-glycoprotein (P-gp/Abcb1) and multidrug resistance-associated proteins (Mrp's/Abcc's) are expressed in the blood-brain barrier (BBB). The aim of this study was to investigate if a bovine endothelial/rat astrocyte in vitro BBB co-culture model displayed polarized transport of known efflux transporter substrates. The co-culture model displayed low mannitol permeabilities of 0.95 ± 0.1 · 10(-6) cm·s(-1) and high transendothelial electrical resistances of 1,177 ± 101 Ω·cm(2). Bidirectional transport studies with (3)H-digoxin, (3)H-estrone-3-sulphate and (3)H-etoposide revealed polarized transport favouring the brain-to-blood direction for all substrates. Steady state efflux ratios of 2.5 ± 0.2 for digoxin, 4.4 ± 0.5 for estrone-3-sulphate and 2.4 ± 0.1 for etoposide were observed. These were reduced to 1.1 ± 0.08, 1.4 ± 0.2 and 1.5 ± 0.1, by addition of verapamil (digoxin), Ko143 (estrone-3-sulphate) or zosuquidar + reversan (etoposide), respectively. Brain-to-blood permeability of all substrates was investigated in the presence of the efflux transporter inhibitors verapamil, Ko143, zosuquidar, reversan and MK 571 alone or in combinations. Digoxin was mainly transported via P-gp, estrone-3-sulphate via Bcrp and Mrp's and etoposide via P-gp and Mrp's. The expression of P-gp, Bcrp and Mrp-1 was confirmed using immunocytochemistry. The findings indicate that P-gp, Bcrp and at least one isoform of Mrp are functionally expressed in our bovine/rat co-culture model and that the model is suitable for investigations of small molecule transport.

Hyperosmotic stress regulates the distribution and stability of myocardin-related transcription factor, a key modulator of the cytoskeleton.

Hyperosmotic stress initiates several adaptive responses, including the remodeling of the cytoskeleton. Besides maintaining structural integrity, the cytoskeleton has emerged as an important regulator of gene transcription. Myocardin-related transcription factor (MRTF), an actin-regulated coactivator of serum response factor, is a major link between the actin skeleton and transcriptional control. We therefore investigated whether MRTF is regulated by hyperosmotic stress. Here we show that hypertonicity induces robust, rapid, and transient translocation of MRTF from the cytosol to the nucleus in kidney tubular cells. We found that the hyperosmolarity-triggered MRTF translocation is mediated by the RhoA/Rho kinase (ROK) pathway. Moreover, the Rho guanine nucleotide exchange factor GEF-H1 is activated by hyperosmotic stress, and it is a key contributor to the ensuing RhoA activation and MRTF translocation, since siRNA-mediated GEF-H1 downregulation suppresses these responses. While the osmotically induced RhoA activation promotes nuclear MRTF accumulation, the concomitant activation of p38 MAP kinase mitigates this effect. Moderate hyperosmotic stress (600 mosM) drives MRTF-dependent transcription through the cis-element CArG box. Silencing or pharmacological inhibition of MRTF prevents the osmotic stimulation of CArG-dependent transcription and renders the cells susceptible to osmotic shock-induced structural damage. Interestingly, strong hyperosmolarity promotes proteasomal degradation of MRTF, concomitant with apoptosis. Thus, MRTF is an osmosensitive and osmoprotective transcription factor, whose intracellular distribution is regulated by the GEF-H1/RhoA/ROK and p38 pathways. However, strong osmotic stress destabilizes MRTF, concomitant with apoptosis, implying that hyperosmotically induced cell death takes precedence over epithelial-myofibroblast transition, a potential consequence of MRTF-mediated phenotypic reprogramming.

HER2 as a Prognostic Marker in Gastric Cancer - A Systematic Analysis of Data from the Literature.

Through the recent conduct of the ToGA trial, HER2 has shown to be predictive for the treatment with trastuzumab in advanced gastric and gastro-oesophageal cancer. When it comes to the prognostic properties the situation is different. Despite the fact that it is more than 20 years ago since the first studies demonstrating an association between a positive HER2 status and poor prognosis were published the issue is still controversial. In this current systematic review a large number of studies on HER2 and gastric cancer have been reviewed. The studies included in this review should fulfill the following two criteria. First criterion: The number of patients in each study should be ≥ 100, and the HER2 status should have been determined either by immunohistochemistry (IHC) or in situ hybridization (ISH). Second criterion: The selected articles should include an analysis of the association between the HER2 status and survival or relevant clinicopathological characteristics. Forty-two publications with a total of 12,749 patients fulfilled the two criteria and were reviewed in detail. The majority of the publications (71%) showed that a HER2-postive status measured either by IHC or ISH was associated with poor survival and/or clinicopathological characteristics, such as serosal invasion, lymph node metastases, disease stage, or distant metastases. Based on the current analysis a clear trend towards a potential role for HER2 as a negative prognostics factor in gastric cancer was shown, suggesting that HER2 overexpression and/or amplification is a molecular abnormality that might be linked to the development of gastric cancer.