Disrupting EGFR-HER2 Transactivation by Pertuzumab in HER2-Positive Cancer: Quantitative Analysis Reveals EGFR Signal Input as Potential Predictor of Therapeutic Outcome.

Pertuzumab (Perjeta®), a humanized antibody binding to the dimerization arm of HER2 (Human epidermal growth factor receptor-2), has failed as a monotherapy agent in HER2 overexpressing malignancies. Since the molecular interaction of HER2 with ligand-bound EGFR (epidermal growth factor receptor) has been implied in mitogenic signaling and malignant proliferation, we hypothesized that this interaction, rather than HER2 expression and oligomerization alone, could be a potential molecular target and predictor of the efficacy of pertuzumab treatment. Therefore, we investigated static and dynamic interactions between HER2 and EGFR molecules upon EGF stimulus in the presence and absence of pertuzumab in HER2+ EGFR+ SK-BR-3 breast tumor cells using Förster resonance energy transfer (FRET) microscopy and fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS). The consequential activation of signaling and changes in cell proliferation were measured by Western blotting and MTT assay. The autocorrelation functions of HER2 diffusion were best fitted by a three-component model corrected for triplet formation, and among these components the slowly diffusing membrane component revealed aggregation induced by EGFR ligand binding, as evidenced by photon-counting histograms and co-diffusing fractions. This aggregation has efficiently been prevented by pertuzumab treatment, which also inhibited the post-stimulus interaction of EGFR and HER2, as monitored by changes in FRET efficiency. Overall, the data demonstrated that pertuzumab, by hindering post-stimulus interaction between EGFR and HER2, inhibits EGFR-evoked HER2 aggregation and phosphorylation and leads to a dose-dependent decrease in cell proliferation, particularly when higher amounts of EGF are present. Consequently, we propose that EGFR expression on HER2-positive tumors could be taken into consideration as a potential biomarker when predicting the outcome of pertuzumab treatment.

The Effect of Fluorophore Conjugation on Antibody Affinity and the Photophysical Properties of Dyes.

Because the degree of labeling (DOL) of cell-bound antibodies, often required in quantitative fluorescence measurements, is largely unknown, we investigated the effect of labeling with two different fluorophores (AlexaFluor546, AlexaFluor647) in a systematic way using antibody stock solutions with different DOLs. Here, we show that the mean DOL of the cell-bound antibody fraction is lower than that of the stock using single molecule fluorescence measurements. The effect is so pronounced that the mean DOL levels off at approximately two fluorophores/IgG for some antibodies. We developed a method for comparing the average DOL of antibody stocks to that of the isolated, cell-bound fraction based on fluorescence anisotropy measurements confirming the aforementioned conclusions. We created a model in which individual antibody species with different DOLs, present in an antibody stock solution, were assumed to have distinct affinities and quantum yields. The model calculations confirmed that a calibration curve constructed from the anisotropy of antibody stocks can be used for determining the DOL of the bound fraction. The fluorescence intensity of the cell-bound antibody fractions and of the antibody stocks exhibited distinctly different dependence on the DOL. The behavior of the two dyes was systematically different in this respect. Fitting of the model to these data revealed that labeling with each dye affects quantum yield and antibody affinity differentially. These measurements also implied that fluorophores in multiply labeled antibodies exhibit self-quenching and lead to decreased antibody affinity, conclusions directly confirmed by steady-state intensity measurements and competitive binding assays. Although the fluorescence lifetime of antibodies labeled with multiple fluorophores decreased, the magnitude of this change was not sufficient to account for self-quenching indicating that both dynamic and static quenching processes occur involving H-aggregate formation. Our results reveal multiple effects of fluorophore conjugation, which must not be overlooked in quantitative cell biological measurements.

B" and C subunits of PP2A regulate the levels of reactive oxygen species and superoxide dismutase activities in Arabidopsis.

The serine-threonine protein phosphatases PP2A regulate many cellular processes, however their role in oxidative stress responses and defence is less known. We show the involvement of its C (catalytic) and B" (a regulatory) subunits. The c3c4 (C subunit) and fass (B") subunit mutants and Col wt of Arabidopsis were used. Controls and treatments with the PP2A inhibitor microcystin-LR (MCY-LR) and reactive oxygen species (ROS) inducer diquat (DQ) were employed. ROS levels of primary roots were largely genotype dependent and both C and B" subunit mutants had increased sensitivity to MCY-LR and DQ indicating the involvement of these subunits in oxidative stress induction. Superoxide dismutases (SOD), mainly the Cu/Zn-SOD isoform, as key enzymes involved in ROS scavenging are also showing altered (mostly increased) activities in both c3c4 and fass mutants and have opposite relations to ROS induction. This indicates that the two types of subunits involved have partially different regulatory roles. In relation to this, control and MCY-LR/DQ treated B" subunit mutants were proven to have altered levels of phosphorylation of histone H2AX. γH2AX, the phosphorylated form indicates double stranded DNA damage during oxidative stress. Overall we point out the probable pivotal role of several PP2A subunits in the regulation of oxidative stress responses in plants and pave the way for future research to reveal the signaling pathways involved.

Macrophages engulf apoptotic and primary necrotic thymocytes through similar phosphatidylserine-dependent mechanisms.

One of the major roles of professional phagocytes is the removal of dead cells in the body. We know less about the clearance of necrotic cells than apoptotic cell phagocytosis, despite the fact that both types of dead cells need to be cleared together and necrotic cells appear often in pathological settings. In the present study, we examined phagocytosis of heat- or H2O2-killed necrotic and apoptotic thymocytes by mouse bone marrow-derived macrophages (BMDMs) in vitro and found that the two cell types are engulfed at equal efficiency and compete with each other when added together to BMDMs. Phagocytosis of both apoptotic and necrotic thymocytes was decreased by (a) blocking phosphatidylserine on the surface of dying cells; (b) inhibition of Mer tyrosine kinase, Tim-4, integrin ß3 receptor signaling, or Ras-related C3 botulinum toxin substrate 1 activity; or (c) using BMDMs deficient for transglutaminase 2. Stimulation of liver X, retinoid X, retinoic acid or glucocorticoid nuclear receptors in BMDMs enhanced not only apoptotic, but also necrotic cell uptake. Electron microscopic analysis of the engulfment process revealed that the morphology of phagosomes and the phagocytic cup formed during the uptake of dying thymocytes is similar for apoptotic and necrotic cells. Our data indicate that apoptotic and necrotic cells are cleared via the same mechanisms, and removal of necrotic cells in vivo can be facilitated by molecules known to enhance the uptake of apoptotic cells.

Retinol Saturase Knock-Out Mice are Characterized by Impaired Clearance of Apoptotic Cells and Develop Mild Autoimmunity.

Apoptosis and the proper clearance of apoptotic cells play a central role in maintaining tissue homeostasis. Previous work in our laboratory has shown that when a high number of cells enters apoptosis in a tissue, the macrophages that engulf them produce retinoids to enhance their own phagocytic capacity by upregulating several phagocytic genes. Our data indicated that these retinoids might be dihydroretinoids, which are products of the retinol saturase (RetSat) pathway. In the present study, the efferocytosis of RetSat-null mice was investigated. We show that among the retinoid-sensitive phagocytic genes, only transglutaminase 2 responded in macrophages and in differentiating monocytes to dihydroretinol. Administration of dihydroretinol did not affect the expression of the tested genes differently between differentiating wild type and RetSat-null monocytes, despite the fact that the expression of RetSat was induced. However, in the absence of RetSat, the expression of numerous differentiation-related genes was altered. Among these, impaired production of MFG-E8, a protein that bridges apoptotic cells to the αvß3/ß5 integrin receptors of macrophages, resulted in impaired efferocytosis, very likely causing the development of mild autoimmunity in aged female mice. Our data indicate that RetSat affects monocyte/macrophage differentiation independently of its capability to produce dihydroretinol at this stage.

Flow Cytometric FRET Analysis of Protein Interactions.

In the past decades, investigation of protein-protein interactions in situ in living or intact cells has gained expanding importance as structure/function relationships proposed from bulk biochemistry and molecular modeling experiments required confirmation at the cellular level. Förster (fluorescence) resonance energy transfer (FRET)-based methods are excellent tools for determining proximity and supramolecular organization of biomolecules at the cell surface or inside the cell. This could well be the basis for the increasing popularity of FRET. In fact, the number of publications exploiting FRET has exploded since the turn of the millennium. Interestingly, most applications are microscope-based, and only a fraction employs flow cytometry, even though the latter offers great statistical power owed to the potentially huge number of individually measured cells. However, with the increased availability of multi-laser flow cytometers, strategies to obtain absolute FRET efficiencies can now be relatively facilely implemented. In this chapter, we intend to provide generally useable protocols for measuring FRET in flow cytometry. After a concise theoretical introduction, recipes are provided for successful labeling techniques and measurement approaches. The simple, quenching-based population-level measurement, the classic ratiometric, intensity-based technique providing cell-by-cell actual FRET efficiencies, and a more advanced version of the latter, allowing for cell-by-cell autofluorescence correction are described. An Excel macro pre-loaded with spectral data of the most commonly used fluorophores is also provided for easy calculation of average FRET efficiencies. Finally, points of caution are given to help design proper experiments and critically interpret the results.

Cultivation of Human Oral Mucosal Explants on Contact Lenses.

Purpose/Aim: Autologous cultivated oral mucosal (OM) epithelial transplantation has been successfully used as corneal epithelial replacement in bilateral limbal stem cell deficiency. Recently, lotrafilcon A contact lens (CL) surface was described as a suitable carrier for cultured stem cells in corneal epithelial transplantation. Our aim was to establish explant cultures from human OM on CL carriers that are free of animal-derived materials and feeder cells. MATERIALS AND METHODS: Human cadaveric 2 mm OM explants were sutured onto CL surfaces and cultivated with fetal calf serum (FCS) or human serum (HS) supplemented culture medium without feeder cells. Confluent cultures were harvested and evaluated morphologically with hematoxylin and eosin stain and with immunofluorescence microscopy for the presence of p63, vimentin and cytokeratins (CK) 3, 4, 13 and 14. RESULTS: Confluent cell sheets covering the whole CL surface were produced from OM explants after 2 weeks of culture with HS and after 3 weeks with FCS. A basal layer consisted of small, vimentin, p63 and CK14 positive putative stem/progenitor cells, which were present in the whole cell sheet. Large, CK3, CK4 and CK13 positive, differentiated cells appeared to spread above this confluent layer. CONCLUSIONS: We have established an animal-free culture system from human OM explants on CL surface. The cultured OM sheets contain large numbers of putative stem cells including limbal-like CK3 and CK14 positive cells. This method can be adapted to good manufacturing practice (GMP) conditions and has, therefore, great potential for clinical use.

Cell confluence induces switching from proliferation to migratory signaling by site-selective phosphorylation of PDGF receptors on lipid raft platforms.

Platelet derived growth factor receptors (PDGFR) play an important role in tumor pathogenesis and are frequently overexpressed in glioblastoma. Earlier we have shown that only confluent glioblastoma cell cultures exhibit a biphasic calcium transient upon PDGF stimulation. Here, we examined how the change in cell density leads to differential cellular responses to the same PDGF stimulus. PDGF beta receptors and their specific phosphotyrosine residues were fluorescently co-labeled on A172 and T98G glioblastoma cells. The distribution in cell membrane microdomains (lipid rafts) and the phosphorylation state of PDGFR was measured by confocal microscopy and quantitated by digital image processing. Corresponding bulk data were obtained by Western blotting. Activation of relevant downstream signaling pathways was assessed by immunofluorescence in confocal microscopy and by Western blot analysis. Functional outcomes were confirmed with bulk and single cell proliferation assays and motility measurements. In non-confluent (sparse) cultures PDGF-BB stimulation significantly increased phosphorylation of Tyr716 specific for the Ras/MAPK pathway and Tyr751 specific for the phosphoinositide 3-kinase/Akt pathway. As cell monolayers reached confluence, Tyr771 and Tyr1021 were the prominently phosphorylated residues. Tyr771 serves as adaptor for Ras-GAP, which inactivates the MAPK pathway, and Tyr1021 feeds into the phospholipase C-gamma/PKC pathway. Coherent with this, MAPK phosphorylation, Ki-67 positivity and proliferation dominated in dispersed cells, and could be abolished with inhibitors of the MAPK pathway. At the same time, RhoA activation, redistribution of cortactin to leading edges, and increased motility were the prominent output features in confluent cultures. Importantly, the stimulus-evoked confluence-specific changes in the phosphorylation of tyrosine residues occurred mainly in GM1-rich lipid microdomains (rafts). These observations suggest that the same stimulus is able to promote distinctly relevant signaling outputs through a confluence dependent, lipid raft-based regulatory mechanism. In particular, cell division and survival in sparse cultures and inhibition of proliferation and promotion of migration in confluent monolayers. In our model, the ability to switch the final output of the same stimulus as a function of cell density could be a key to the balance of proliferation and invasion in malignant glioblastoma.