Adoptive transfer of T cells that are gene engineered to express a defined TCR represents a feasible and promising therapy for patients with tumors. However, TCR gene therapy is hindered by the transient presence and effectiveness of transferred T cells, which are anticipated to be improved by adequate T cell costimulation. In this article, we report the identification and characterization of a novel two-chain TCR linked to CD28 and CD3? (i.e., TCR:28?). This modified TCR demonstrates enhanced binding of peptide-MHC and mediates enhanced T cell function following stimulation with peptide compared with wild-type TCR. Surface expression of TCR:28? depends on the transmembrane domain of CD28, whereas T cell functions depend on the intracellular domains of both CD28 and CD3?, with IL-2 production showing dependency on CD28:LCK binding. TCR:28?, but not wild-type TCR, induces detectable immune synapses in primary human T cells, and such immune synapses show significantly enhanced accumulation of TCR transgenes and markers of early TCR signaling, such as phosphorylated LCK and ERK. Importantly, TCR:28? does not show signs of off-target recognition, as evidenced by lack of TCR mispairing, as well as preserved specificity. Notably, when testing TCR:28? in immune-competent mice, we observed a drastic increase in T cell survival, which was accompanied by regression of large melanomas with limited recurrence. Our data argue that TCR transgenes that contain CD28, and, thereby, may provide T cell costimulation in an immune-suppressive environment, represent candidate receptors to treat patients with tumors.
Cluster of differentiation 1 (CD1) represents a family of proteins which is involved in lipid-based antigen presentation. Primarily, antigen presenting cells, like B cells, express CD1 proteins. Here, we examined the cell-surface distribution of CD1d, a subtype of CD1 receptors, on B lymphocytes.
Since the administration of synthetic medicines is associated with drug resistance and undesired side effects, utilization of natural compounds could be an alternative and complementary modality to inhibit or prevent the development of tumors. Epigallocatechin 3-O-gallate (EGCG, 1), the major flavan component of green tea, and genistein (2), a soy isoflavonoid, are known to have chemopreventive and chemotherapeutic effects against cancer. This study demonstrated that both flavonoids inhibit cell proliferation, an effect enhanced under serum-free conditions. Compound 1, but not 2, induced downregulation of ErbB1 and ErbB2 in mammary and epidermoid carcinoma cells, and its inhibitory effect on cell viability was mediated by the 67 kDa laminin receptor (67LR). While 1 was superior in inducing cell death, 2 was more efficient in arresting the tumor cells in the G2/M phase. Furthermore, number and brightness analysis revealed that 1 decreased the homoclustering of a lipid raft marker, glycosylphosphatidylinositol-anchored GFP, and it also reduced the co-localization between lipid rafts and 67LR. The main conclusion made is that the primary target of 1 may be the lipid raft component of the plasma membrane followed by secondary changes in the expression of ErbB proteins. Compound 2, on the other hand, must have other unidentified targets.
The mechanism of action of elisidepsin (PM02734, Irvalec®) is assumed to involve membrane permeabilization via attacking lipid rafts and hydroxylated lipids. Here we investigate the role of hypoxia in the mechanism of action of elisidepsin. Culturing under hypoxic conditions increased the half-maximal inhibitory concentration and decreased the drugs binding to almost all cell lines which was reversed by incubation of cells with 2-hydroxy palmitic acid. The expression of fatty acid 2-hydroxylase was strongly correlated with the efficiency of the drug and inversely correlated with the effect of hypoxia. Number and brightness analysis and fluorescence anisotropy experiments showed that hypoxia decreased the clustering of lipid rafts and altered the structure of the plasma membrane. Although the binding of elisidepsin to the membrane is non-cooperative, its membrane permeabilizing effect is characterized by a Hill coefficient of ~3.3. The latter finding is in agreement with elisidepsin-induced clusters of lipid raft-anchored GFP visualized by confocal microscopy. We propose that the concentration of elisidepsin needs to reach a critical level in the membrane above which elisidepsin induces the disruption of the cell membrane. Testing for tumor hypoxia or the density of hydroxylated lipids could be an interesting strategy to increase the efficiency of elisidepsin.
Laser scanning cytometry (LSC) is a slide-based technique combining advantages of flow and image cytometry: automated, high-throughput detection of optical signals with subcellular resolution. Fluorescence resonance energy transfer (FRET) is a spectroscopic method often used for studying molecular interactions and molecular distances. FRET has been measured by various microscopic and flow cytometric techniques. We have developed a protocol for a commercial LSC instrument to measure FRET on a cell-by-cell or pixel-by-pixel basis on large cell populations, which adds a new modality to the use of LSC. As a reference sample for FRET, we used a fusion protein of a single donor and acceptor (ECFP-EYFP connected by a seven-amino acid linker) expressed in HeLa cells. The FRET efficiency of this sample was determined via acceptor photobleaching and used as a reference value for ratiometric FRET measurements. Using this standard allowed the precise determination of an important parameter (the alpha factor, characterizing the relative signal strengths from a single donor and acceptor molecule), which is indispensable for quantitative FRET calculations in real samples expressing donor and acceptor molecules at variable ratios. We worked out a protocol for the identification of adherent, healthy, double-positive cells based on light-loss and fluorescence parameters, and applied ratiometric FRET equations to calculate FRET efficiencies in a semi-automated fashion. To test our protocol, we measured the FRET efficiency between Fos-ECFP and Jun-EYFP transcription factors by LSC, as well as by confocal microscopy and flow cytometry, all yielding nearly identical results. Our procedure allows for accurate FRET measurements and can be applied to the fast screening of protein interactions. A pipeline exemplifying the gating and FRET analysis procedure using the CellProfiler software has been made accessible at our web site.
Treatment of astrocytoma is frequently hampered by radioresistance of the tumor. In addition to overexpression of ErbB1/EGFR, functional crosstalk between receptor tyrosine kinases and cell adhesion molecules may also contribute to therapy resistance.
Proteins are the major targets of drug discovery and many of the new drugs are designed to exert their effect by disrupting protein-protein interactions. Validation of the inhibition of molecular interactions is generally done by biochemical methods, however, these are often not feasible when the interaction is not stable enough. Fluorescence resonance energy transfer (FRET) is an excellent tool for determining direct molecular interactions between two molecules in the cell membrane or inside cells in their natural state. Although originally established as a flow cytometric approach, FRET has been adapted for microscopy, allowing for analysis of sub-cellular co-localization at the single cell level. In this chapter, we provide theoretical introduction to the phenomenon of FRET, and a protocol - including labeling techniques, measurement, and evaluation of microscopy images - of the simplest microscopic FRET approach, acceptor photobleaching FRET. This technique is generally usable for studying protein interactions and requires only a standard confocal laser scanning microscope. To demonstrate the value of image based FRET for testing pharmacological disruption of protein-protein interactions, we show how inhibition of the hetero-dimerization of ErbB2 and ErbB1 by the humanized monoclonal antibody pertuzumab can be validated using this technique.
A frequently used method for viewing protein interactions and conformation, Förster (fluorescence) resonance energy transfer (FRET), has traditionally been restricted to two fluorophores. Lately, several methods have been introduced to expand FRET methods to three species. We present a method that allows the determination of FRET efficiency in three-dye systems on a flow cytometer. TripleFRET accurately reproduces energy transfer efficiency values measured in two-dye systems, and it can indicate the presence of trimeric complexes, which is not possible with conventional FRET methods. We also discuss the interpretation of energy transfer values obtained with tripleFRET in relation to spatial distribution of labeled molecules, specifically addressing the limitations of using total energy transfer to determine molecular distance.
Bare lymphocyte syndrome (BLS) is a rare immunodeficiency disorder manifested by the partial or complete disappearance of major histocompatibility complex (MHC) proteins from the surface of the cells. Based on this specific feature, it is categorized into three different types depending on which type of MHC protein is affected. These proteins are mainly involved in generating the effective immune responses by differentiating self from non-self antigens through a process referred to as antigen presentation. Investigations on BLS have immensely contributed to our understanding of the transcriptional regulation of these molecules and have led to the discovery of several important proteins of the antigen presentation pathway. Reviews on this subject consistently project type II BLS, MHC II deficiency as BLS syndrome, although literatures document cases of other types of BLS too. Therefore, in this article, we have assembled information on the BLS syndrome to produce a systematic narration while emphasizing the importance of BLS system in studying various aspects of immune biology.
Investigation of protein-protein interactions in situ in living or intact cells gains expanding importance as structure/function relationships proposed from bulk biochemistry and molecular modeling experiments require demonstration at the cellular level. 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 doubled in the past 5 years. In this chapter, we intend to provide a generally useable protocol 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. Finally, points of caution are given to help design proper experiments and critically interpret the results.
Elisidepsin is a marine-derived anti-tumor agent with unique mechanism of action. It has been suggested to induce necrosis associated with severe membrane damage. Since indirect evidence points to the involvement of ErbB receptor tyrosine kinases and lipid rafts in the mechanism of action of elisidepsin, we investigated the effect of the drug on the distribution of ErbB proteins and systematically compared the elisidepsin sensitivity of cell lines overexpressing ErbB receptors. Stable expression of a single member of the ErbB family (ErbB1-3) or co-transfection of ErbB2 and ErbB3 did not modify the elisidepsin sensitivity of CHO and A431 cells. However, elisidepsin induced the redistribution of ErbB3 and two GPI-anchored proteins (transfected GPI-anchored eGFP and placental alkaline phosphatase) from the plasma membrane to intracellular vesicles without comparable effects on ErbB1 and ErbB2. Elisidepsin increased the binding of a conformational sensitive anti-ErbB3 antibody without modifying the binding of other ErbB2 or ErbB3 antibodies, and it decreased the homoassociation of both ErbB2 and ErbB3. We also found that elisidepsin decreased the fluorescence anisotropy of a membrane specific fluorescent probe and induced a blue shift in the emission spectrum of Laurdan pointing to significant changes in the order of the plasma membrane possibly associated with the formation of liquid ordered domains. Although the distribution of ErbB proteins is preferentially altered by elisidepsin, our data question their role in determining sensitivity to the drug. We assume that induction of liquid ordered domains is the primary action of elisidepsin leading to all the other observed changes.
T-cell receptors (TCRs) can be genetically modified to improve gene-engineered T-cell responses, a strategy considered critical for the success of clinical TCR gene therapy to treat cancers. TCR:?, which is a heterodimer of TCR? and ? chains each coupled to complete human CD3?, overcomes issues of mis-pairing with endogenous TCR chains, shows high surface expression and mediates antigen-specific T-cell functions in vitro. In the current study, we further characterized TCR:? in gene-engineered T cells and assessed whether this receptor is able to interact with surface molecules and drive correct synapse formation in Jurkat T cells. The results showed that TCR:? mediates the formation of synaptic areas with antigen-positive target cells, interacts closely with CD8? and MHC class I (MHCI), and co-localizes with CD28, CD45 and lipid rafts, similar to WT TCR. TCR:? did not closely associate with endogenous CD3?, despite its co-presence in immune synapses, and TCR:? showed enhanced synaptic accumulation in T cells negative for surface-expressed TCR molecules. Notably, synaptic TCR:? demonstrated lowered densities when compared with TCR in dual TCR T cells, a phenomenon that was related to both extracellular and intracellular CD3? domains present in the TCR:? molecule and responsible for enlarged synapse areas.
Both fluorescence resonance energy transfer (FRET) and proximity ligation assay (PLA) are techniques used in the investigation of protein interactions but the latter has not been evaluated in a systematic way, prompting us to compare their performance quantitatively. Proteins were labeled with oligonucleotide- or fluorophore-conjugated antibodies and their proximity was analyzed by flow cytometry in order to obtain statistically robust data. Both intermolecular and intramolecular PLA signals reached saturation at high expression levels. At the same time, the FRET efficiency was independent of, while the FRET signal exhibited a strict linear correlation with the expression levels of proteins. When the density of oligonucleotide- and fluorophore-conjugated antibodies was systematically changed by competition with unlabeled antibodies the FRET signal was linearly proportional to the amount of bound fluorophore-tagged antibodies, whereas the PLA signal was again saturated. The saturation phenomenon in PLA could not be eliminated by decreasing the duration of the rolling circle amplification reaction. Our data imply that PLA is a semiquantitative measure of protein colocalizations due to non-linear effects in the reaction and that caution should be exercised when interpreting PLA data in a quantitative way.
TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) can selectively trigger apoptosis in various cancer cell types. However, many cancer cells are resistant to death receptor-mediated apoptosis. Combination therapy with platinum complexes may affect TRAIL-induced signaling via modulation of various steps in apoptotic pathways. Here, we show that cisplatin or a more potent platinum(IV) complex LA-12 used in 20-fold lower concentration enhanced killing effects of TRAIL in human colon and prostate cancer cell lines via stimulation of caspase activity and overall apoptosis. Both platinum complexes increased DR5 surface expression in colon cancer cells. Small interfering RNA-mediated DR5 silencing rescued cells from sensitizing effects of platinum drugs on TRAIL-induced caspase-8 activation and apoptosis, showing the functional importance of DR5 in the effects observed. In addition, both cisplatin and LA-12 triggered the relocalization of DR4 and DR5 receptors to lipid rafts and accelerated internalization of TRAIL, which may also affect TRAIL signaling. Collectively, modulations of the initial steps of the extrinsic apoptotic pathway at the level of DR5 and plasma membrane are important for sensitization of colon and prostate cancer cells to TRAIL-induced apoptosis mediated by LA-12 and cisplatin.
Fluorescence resonance energy transfer (FRET) is a strongly distance-dependent process between a donor and an acceptor molecule, which can be used for sensitive distance measurements and characterization of molecular interactions at the nanometer level. The original mathematical description of this process, however, is only valid for the interaction of one donor with one acceptor. This criterion is not always met, especially in biological systems, where multiple structures can interact simultaneously, often making distance estimations based on transfer efficiency values error-prone. Herein we investigate how the interaction of multiple acceptors and donors influences the transfer efficiency value in an intramolecular cellular FRET system by manipulating the fluorophore/protein ratio of the fluorophore-conjugated antibodies. We show that the labeling ratio of the acceptor has the largest influence on measured transfer efficiency and decreasing or increasing the acceptor labeling ratio can be utilized to manipulate the FRET response of the acceptor-donor pair and therefore is a tool for optimizing sensitivity of FRET measurements.
Patients with unexplained infertility may have fertilization problems. Split fertilization (ICSI and conventional IVF on sibling oocytes) is often used to avoid poor fertilization. Our aim was to assess the ability of hyaluronic acid binding (HA-binding) assay to predict spontaneous fertilization during IVF.
Classical theory states that ligand binding induces the dimerization of ErbB proteins, leading to their activation. Although we and other investigators have shown the existence of preformed homoclusters of ErbB receptors and analyzed their composition, the stoichiometry of their heteroclusters has not been quantitatively described. Here, we report the development of the fluorescence resonance energy transfer (FRET)-sensitized acceptor bleaching (FSAB) technique to quantitate the ratio of ErbB1 and ErbB2 in their heteroclusters. In FSAB, photolabile acceptors within FRET distance from photostable donors are excited and photobleached by FRET, and the fraction of acceptors that are participating in FRET is determined. In quiescent SKBR-3 breast cancer cells, approximately 35% of ErbB1 and approximately 10% of ErbB2 have been found in heteroclusters. Epidermal growth factor (ligand of ErbB1) increased the fraction of ErbB2 heteroclustering with ErbB1, whereas the ratio of heteroclustered ErbB1 did not change significantly. The fractions of heteroclustered ErbB1 and ErbB2 were independent of their expression levels, indicating that the formation of these clusters is not driven by the law of mass action. In contrast, the FRET efficiency depended on the donor/acceptor ratio as expected. We present a model in which preformed receptor clusters are rearranged upon ligand stimulation, and report that the composition of these clusters can be quantitatively described by the FSAB technique.
Recognition of the heterogeneity of the cell membrane was one of the most important scientific achievements in the last decades. Since coining the term "lipid rafts", continuous development of advanced microscopic and spectroscopic techniques has vastly expanded our view on these cell membrane microdomains that appear to have almost as many faces as researchers that look at them; they are variable in stability, size and composition that can change in a highly dynamic manner both by recruiting and expelling components as well as by coalescing and breaking up into smaller units. They have, however, one common feature: all eukaryotic cells present some variation of lipid rafts. Cells of the immune system are not exception to this, regardless of their lymphoid or myeloid origin their membranes show a domain structure and these domains serve to condense or reject particular transmembrane, GPI-linked and intracellularly membrane-anchored proteins as function requires. Here we provide a concise overview about the various weapons and shields that immune cells concentrate into their rafts, which have come into sight during the past years. The positive and negative regulatory roles of these microdomains are essential both in the functions of innate immunity and processes concatenated in the adaptive immune response.
Fluorescence correlation spectroscopy (FCS) is a fluctuation method established three decades ago, whose application to cellular systems became popular in the last decade. Fluctuations of fluorescence emission are observed from a small, femtoliter to sub-femtoliter, usually confocal volume at high time resolution. A time-dependent autocorrelation function is generated and evaluated to obtain time constants of photophysical and photochemical reactions, as well as of molecular diffusion and in the observation volume. Molecules in various subcellular compartments-including the nucleus, the cytoplasm, and the membrane-can be observed after labeling them with antibodies, ligands, or fluorescent proteins. The anomaly of diffusion, the local concentration, and the average fluorescence per diffusing particle can also be determined, all of which can be characteristic of molecular interactions. A two-color version of FCS, fluorescence cross-correlation spectroscopy, can also be applied to observe co-diffusion, i.e., stable association of two distinct molecular species in their cellular environment.
Tumor cell invasion into the surrounding brain tissue is mainly responsible for the failure of radical surgical resection and successful treatment, with tumor recurrence as microdisseminated disease. Epidermal growth factor receptors (EGFRs), integrins and their ligands in the extracellular matrix (ECM) predominantly participate in the invasion process, including the cell adhesion to the surrounding microenvironment and cell migration. The extent of infiltration of the surrounding brain tissue by malignant tumors strongly depends on the tumor cell type. Malignant gliomas show much more intensive peritumoral invasion than do metastatic tumors. In this study, the mRNA expression of 29 invasion-related molecules (18 cell membrane receptors or receptor subunits (EGFRs and integrins) and 11 ECM components: collagens, laminins and fibronectin) was investigated by quantitative reverse transcriptase-polymerase chain reaction. Fresh frozen human tissue samples from glioblastoma (GBM) and intracerebral bronchial adenocarcinoma metastases (five pieces from each) were evaluated. Significant differences were established in six of the 29 molecules (ErbB1, 2, 3, integrins alpha3, 7 and beta1). To confirm our results at the protein level, immunohistochemical analysis of nine molecules was performed. The staining intensity differed definitely in the case of ErbB1, 2 and integrins alpha3 and beta1. Determining the differences in invasion-related molecules in tumors of different origin can help identify the exact molecular mechanisms that facilitate peritumoral infiltration by glioblastoma cells. These results should allow the selection of target molecules for potential chemotherapeutic agents directed against highly invasive malignant gliomas.
The intensity-based ratiometric FRET (fluorescence resonance energy transfer) method is a powerful technique for following molecular interactions in living cells. Since it is not based on irreversibly destroying the donor or the acceptor fluorophores, the time course of changes in FRET efficiency values can be monitored by this method. ImageJ, a sophisticated software tool for many types of image processing allows users to extend it with programs for various purposes. Implementing intensity-based ratiometric FRET with ImageJ vastly enhances the applicability of the FRET method. We developed an efficient ImageJ plugin, RiFRET, which calculates FRET efficiency on a pixel-by-pixel basis from ratiometric FRET images. It allows the user to correct for channel cross-talk (bleed-through) and to calculate FRET from image stacks, i.e., from 3D data sets. Semiautomatic processing for larger datasets is also included in the program. Furthermore, several options for calibrating FRET efficiency calculations were tested and their applicability to various expression systems is discussed. Although the ratiometric FRET method is widely applied, our plugin is the first freely available software for evaluating such FRET data. The program is user friendly and provides reliable, standardized results.
Although cationic liposome-mediated transfection has become a standard procedure, the mechanistic details of the process are unknown. It has been suggested that endocytic uptake of lipoplexes is efficient, and transfectability is largely determined by later steps. In this article, we stained GM1-enriched membrane microdomains, a subclass of lipid rafts, with subunit B of cholera toxin and correlated transfection efficiency with their density by quantitatively evaluating microscopic images. We found a strong anticorrelation between the density of GM1-enriched membrane microdomains and the efficacy of transfection monitored by measuring the expression level of GFP in different cell lines transfected by lipofection using two different transfection agents. These findings imply that GM1-enriched membrane microdomains interfere with the process of lipofection. The blocked step must be endocytosis since the accumulation of fluorescently labeled plasmids was lower in cells with high content of GM1-enriched membrane microdomains. Such a correlation was not observed in cells transfected by electroporation. By comparing the efficiency of lipofection in several cell lines we found that those with a high density of GM1-enriched membrane microdomains were the most resistant to transfection. We conclude that the inhibition of lipofection by GM1-enriched membrane microdomains is a general rule, and that endocytosis of lipoplexes can be rate limiting in cells with high density of GM1-enriched membrane rafts.
In recent years, an exponentially growing number of studies have focused on identifying cancer stem cells (CSC) in human malignancies. The rare CSCs could be crucial in controlling and curing cancer: through asymmetric division CSCs supposedly drive tumor growth and evade therapy with the help of traits shared with normal stem cells such as quiescence, self-renewal ability, and multidrug resistance pump activity. Here, we give a brief overview of techniques used to confirm the stem cell-like behavior of putative CSCs and discuss markers and methods for identifying, isolating, and culturing them. We touch on the limitations of each marker and why the combined use of CSC markers, in vitro and in vivo assays may still fail to identify all relevant CSC populations. Finally, the various experimental findings supporting and contradicting the CSC hypothesis are summarized. The large number of tumor types thus far with a subpopulation of uniquely tumorigenic and therapy resistant cells suggests that despite the unanswered questions and inconsistencies, the CSC hypothesis has a legitimate role to play in tumor biology. At the same time, experimental evidence supporting the established alternative theory of clonal evolution can be found as well. Therefore, a model that describes cancer initiation and progression should combine elements of clonal evolution and CSC theory.
Cancer stem cell (CSC) biology is a rapidly developing field within cancer research. CSCs are postulated to be a unique cell population exclusively capable of infinite self renewal, multilineage differentiation and with ability to evade conventional cytotoxic cancer therapy. These traits distinguish CSCs from their more differentiated counterparts, which possess only limited or no potential for self renewal and tumor initiation. Therefore, CSCs would be the driving motor of malignant growth and therapy resistance. Accordingly, successful cancer treatment would need to eliminate this highly potent group of cells, since even small residual numbers would suffice to recapitulate the disease after therapy. Putative CSCs has been identified in a broad range of human malignancies and several cell surface markers have been associated with their stem cell phenotype. Despite all efforts, a pure CSC population has not been isolated and often in vitro clonogenic and in vivo tumorigenic potential is found in several cell populations with occasionally contradictory surface marker signatures. Here, we give a brief overview of recent advances in CSC theory, including the signaling pathways in CSCs that also appear crucial for stem cells homeostasis in normal tissues. We discuss evidence for the interaction of CSCs with the stromal tumor environment. Finally, we review the emerging potentially effective CSC-targeted treatment strategies and their future role in therapy.
The progression in bioconjugational chemistry has significantly contributed to the evolution and success of protein biology. Mainly, antibody chemistry has been a subject of intensive study owing to the expansion of research areas warranted by using various derivatives of conjugated antibodies. Three reactive moieties (amine, sulfhydryl and carbohydrate) in the antibodies are chiefly favored for the conjugational purpose. This feature is known for decades, nevertheless, amine based conjugation is still the most preferred strategy despite the appreciation the other two methods receive in conserving the antigen binding affinity (ABA). No single report has been published, according to our knowledge, where these three conjugation strategies were applied to the same fluorophore antibody systems. In this study, we evaluated conjugation yield, time demand and cost efficiency of these conjugation procedures. Our results showed that amine based conjugations was by far the best technique due to its simplicity, rapidity, ease of operation, higher conjugate yield, cheaper cost and potential for larger fluorophore/protein labeling ratio without having much effect in ABA. Furthermore, sulfhydryl labeling clearly excelled in terms of reduced non-specific binding and mild effect in ABA but was usually complicated by an asymmetric antibody reduction due to mercaptoethylamine while carbohydrate oxidation based strategy performed the worst during our experiment.
Although trastuzumab is an efficient drug, primary and acquired resistance is a challenging problem. The authors have previously shown in mouse xenograft experiments that masking ErbB2 by hyaluronan leads to diminished binding of the antibody and consequent resistance. In the current work, they correlated trastuzumab binding with the pericellular density of hyaluronan in ErbB2-overexpressing human breast cancer samples. A method for quantifying the relative binding of trastuzumab was developed involving constant and low-frequency background subtraction, segmenting the image to membrane and background pixels followed by evaluation of trastuzumab fluorescence, normalized with the expression level of ErbB2, only in the membrane. The normalized binding of trastuzumab showed a negative correlation with the pericellular density of hyaluronan (r = -0.52) with the effect being the most pronounced in the extreme cases (i.e., low and high hyaluronan densities predicted strong and weak binding of trastuzumab, respectively). Removal of hyaluronan by hyaluronidase digestion unmasked the trastuzumab binding epitope of ErbB2 demonstrated by a significantly increased normalized binding of the antibody. The results show that the accumulation of pericellular hyaluronan plays a crucial role in masking ErbB2.
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