Use of red, far-red, and near-infrared light in imaging of yeasts and filamentous fungi.

While phototoxicity can be a useful therapeutic modality not only for eliminating malignant cells but also in treating fungal infections, mycologists aiming to observe morphological changes or molecular events in fungi, especially when long observation periods or high light fluxes are warranted, encounter problems owed to altered regulatory pathways or even cell death caused by various photosensing mechanisms. Consequently, the ever expanding repertoire of visible fluorescent protein toolboxes and high-resolution microscopy methods designed to investigate fungi in vitro and in vivo need to comply with an additional requirement: to decrease the unwanted side effects of illumination. In addition to optimizing exposure, an obvious solution is red-shifted illumination, which, however, does not come without compromises. This review summarizes the interactions of fungi with light and the various molecular biology and technology approaches developed for exploring their functions on the molecular, cellular, and in vivo microscopic levels, and outlines the progress towards reducing phototoxicity through applying far-red and near-infrared light. KEY POINTS: • Fungal biological processes alter upon illumination, also under the microscope • Red shifted fluorescent protein toolboxes decrease interference by illumination • Innovations like two-photon, lightsheet, and near IR microscopy reduce phototoxicity.

The multitargeted receptor tyrosine kinase inhibitor sunitinib induces resistance of HER2 positive breast cancer cells to trastuzumab-mediated ADCC.

Despite recent advances in the development of novel personalized therapies, breast cancer continues to challenge physicians with resistance to various advanced therapies. The anticancer action of the anti-HER2 antibody, trastuzumab, involves antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer (NK) cells. Here, we report a repurposing screen of 774 clinically used compounds on NK-cell + trastuzumab-induced killing of JIMT-1 breast cancer cells. Using a calcein-based high-content screening (HCS) assay for the image-based quantitation of ADCC that we have developed and optimized for this purpose, we have found that the multitargeted tyrosine kinase inhibitor sunitinib inhibits ADCC in this model. The cytoprotective effect of sunitinib was also confirmed with two other assays (lactate dehydrogenase release, and electric cell substrate impedance sensing, ECIS). The drug suppressed NK cell activation as indicated by reduced granzyme B deposition on to the target cells and inhibition of interferon-γ production by the NK cells. Moreover, sunitinib induced downregulation of HER2 on the target cells' surface, changed the morphology and increased adherence of the target cells. Moreover, sunitinib also triggered the autophagy pathway (speckled LC3b) as an additional potential underlying mechanism of the cytoprotective effect of the drug. Sunitinib-induced ADCC resistance has been confirmed in a 3D tumor model revealing the prevention of apoptotic cell death (Annexin V staining) in JIMT-1 spheroids co-incubated with NK cells and trastuzumab. In summary, our HCS assay may be suitable for the facile identification of ADCC boosting compounds. Our data urge caution concerning potential combinations of ADCC-based immunotherapies and sunitinib.

Isocyanide Substitution in Acridine Orange Shifts DNA Damage-Mediated Phototoxicity to Permeabilization of the Lysosomal Membrane in Cancer Cells.

In cancer therapy, immunogenic cell death eliminates tumor cells more efficiently than conventional apoptosis. During photodynamic therapy (PDT), some photosensitizer (PS) targeting lysosomes divert apoptosis to the immunologically more relevant necrosis-like cell death. Acridine orange (AO) is a PS targeting lysosome. We synthesized a new compound, 3-N,N-dimethylamino-6-isocyanoacridine (DM), a modified AO, aiming to target lysosomes better. To compare DM and AO, we studied optical properties, toxicity, cell internalization, and phototoxicity. In addition, light-mediated effects were monitored by the recently developed QUINESIn method on nuclei, and membrane stability, morphology, and function of lysosomes utilizing fluorescent probes by imaging cytometry in single cells. DM proved to be a better lysosomal marker at 405 nm excitation and lysed lysosomes more efficiently. AO injured DNA and histones more extensively than DM. Remarkably, DM's optical properties helped visualize shockwaves of nuclear DNA released from cells during the PDT. The asymmetric polar modification of the AO leads to a new compound, DM, which has increased efficacy in targeting and disrupting lysosomes. Suitable AO modification may boost adaptive immune response making PDT more efficient.

From antibodies to living drugs: Quo vadis cancer immunotherapy?

In the last few decades, monoclonal antibodies targeting various receptors and ligands have shown significant advance in cancer therapy. However, still a great percentage of patients experiences tumor relapse despite persistent antigen expression. Immune cell therapy with adoptively transferred modified T cells that express chimeric antigen receptors (CAR) is an engaging option to improve disease outcome. Designer T cells have been applied with remarkable success in the treatment for acute B cell leukemias, yielding unprecedented antitumor activity and significantly improved overall survival. Relying on the success of CAR T cells in leukemias, solid tumors are now emerging potential targets; however, their complexity represents a significant challenge. In preclinical models, CAR T cells recognized and efficiently killed the wide spectrum of tumor xenografts; however, in human clinical trials, limited antitumor efficacy and serious side effects, including cytokine release syndrome, have emerged as potential limitations. The next decade will be an exciting time to further optimize this novel cellular therapeutics to improve effector functions and, at the same time, keep adverse events in check. Moreover, we need to establish whether gene-modified T cells which are yet exclusively used for cancer patients could also be successful in the treatment for other diseases. Here, we provide a concise overview about the transition from monoclonal antibodies to the generation of chimeric antigen receptor T cells. We summarize lessons learned from preclinical models, including our own HER2-positive tumor models, as well as from clinical trials worldwide. We also discuss the challenges we are facing today and outline future prospects.

Cytolytic Activity of CAR T Cells and Maintenance of Their CD4+ Subset Is Critical for Optimal Antitumor Activity in Preclinical Solid Tumor Models.

Correlative studies of clinical studies for hematological malignancies have implicated that less differentiated, CD8+-dominant CAR T cell products have greater antitumor activity. Here, we have investigated whether the differentiation status of CAR T cell products affects their antitumor activity in preclinical models of solid tumors. We explored if different activation/expansion protocols, as well as different co-stimulatory domains in the CAR construct, influence the short- and long-term efficacy of CAR T cells against HER2-positive tumors. We generated T cell products that range from the most differentiated (CD28.z; OKT3-antiCD28/RPMI expansion) to the least differentiated (41BB.z; OKT3-RetroNectin/LymphoONE expansion), as judged by cell surface expression of the differentiation markers CCR7 and CD45RA. While the effect of differentiation status was variable with regard to antigen-specific cytokine production, the most differentiated CD28.z CAR T cell products, which were enriched in effector memory T cells, had the greatest target-specific cytolytic activity in vitro. These products also had a greater proliferative capacity and maintained CD4+ T cells upon repeated stimulation in vitro. In vivo, differentiated CD28.z CAR T cells also had the greatest antitumor activity, resulting in complete response. Our results highlight that it is critical to optimize CAR T cell production and that optimal product characteristics might depend on the targeted antigen and/or cancer.

A Small Number of HER2 Redirected CAR T Cells Significantly Improves Immune Response of Adoptively Transferred Mouse Lymphocytes against Human Breast Cancer Xenografts.

HER2 positive JIMT-1 breast tumors are resistant to trastuzumab treatment in vitro and develop resistance to trastuzumab in vivo in SCID mice. We explored whether these resistant tumors could still be eliminated by T cells redirected by a second-generation chimeric antigen receptor (CAR) containing a CD28 costimulatory domain and targeting HER2 with a trastuzumab-derived scFv. In vitro, T cells engineered with this HER2 specific CAR recognized HER2 positive target cells as judged by cytokine production and cytolytic activity. In vivo, the administration of trastuzumab twice weekly had no effect on the growth of JIMT-1 xenografts in SCID mice. At the same time, a single dose of 2.5 million T cells from congenic mice exhibited a moderate xenoimmune response and even stable disease in some cases. In contrast, when the same dose contained 7% (175,000) CAR T cells, complete remission was achieved in 57 days. Even a reduced dose of 250,000 T cells, including only 17,500 CAR T cells, yielded complete remission, although it needed nearly twice the time. We conclude that even a small number of CAR T lymphocytes can evoke a robust anti-tumor response against an antibody resistant xenograft by focusing the activity of xenogenic T cells. This observation may have significance for optimizing the dose of CAR T cells in the therapy of solid tumors.

Alleviation of Multidrug Resistance by Flavonoid and Non-Flavonoid Compounds in Breast, Lung, Colorectal and Prostate Cancer.

The aim of the manuscript is to discuss the influence of plant polyphenols in overcoming multidrug resistance in four types of solid cancers (breast, colorectal, lung and prostate cancer). Effective treatment requires the use of multiple toxic chemotherapeutic drugs with different properties and targets. However, a major cause of cancer treatment failure and metastasis is the development of multidrug resistance. Potential mechanisms of multidrug resistance include increase of drug efflux, drug inactivation, detoxification mechanisms, modification of drug target, inhibition of cell death, involvement of cancer stem cells, dysregulation of miRNAs activity, epigenetic variations, imbalance of DNA damage/repair processes, tumor heterogeneity, tumor microenvironment, epithelial to mesenchymal transition and modulation of reactive oxygen species. Taking into consideration that synthetic multidrug resistance agents have failed to demonstrate significant survival benefits in patients with different types of cancer, recent research have focused on beneficial effects of natural compounds. Several phenolic compounds (flavones, phenolcarboxylic acids, ellagitannins, stilbens, lignans, curcumin, etc.) act as chemopreventive agents due to their antioxidant capacity, inhibition of proliferation, survival, angiogenesis, and metastasis, modulation of immune and inflammatory responses or inactivation of pro-carcinogens. Moreover, preclinical and clinical studies revealed that these compounds prevent multidrug resistance in cancer by modulating different pathways. Additional research is needed regarding the role of phenolic compounds in the prevention of multidrug resistance in different types of cancer.

Homo- and Heteroassociations Drive Activation of ErbB3.

Dimerization or the formation of higher-order oligomers is required for the activation of ErbB receptor tyrosine kinases. The heregulin (HRG) receptor, ErbB3, must heterodimerize with other members of the family, preferentially ErbB2, to form a functional signal transducing complex. Here, we applied single molecule imaging capable of detecting long-lived and mobile associations to measure their stoichiometry and mobility and analyzed data from experiments globally, taking the different lateral mobility of monomeric and dimeric molecular species into account. Although ErbB3 was largely monomeric in the absence of stimulation and ErbB2 co-expression, a small fraction was present as constitutive homodimers exhibiting a ∼40% lower mobility than monomers. HRG stimulation increased the homodimeric fraction of ErbB3 significantly and reduced the mobility of homodimers fourfold compared to constitutive homodimers. Expression of ErbB2 elevated the homodimeric fraction of ErbB3 even in unstimulated cells and induced a ∼2-fold reduction in the lateral mobility of ErbB3 homodimers. The mobility of ErbB2 was significantly lower than that of ErbB3, and HRG induced a less pronounced decrease in the diffusion coefficient of all ErbB2 molecules and ErbB3/ErbB2 heterodimers than in the mobility of ErbB3. The slower diffusion of ErbB2 compared to ErbB3 was abolished by depolymerizing actin filaments, whereas ErbB2 expression induced a substantial rearrangement of microfilaments, implying a bidirectional interaction between ErbB2 and actin. HRG stimulation of cells co-expressing ErbB3 and ErbB2 led to the formation of ErbB3 homodimers and ErbB3/ErbB2 heterodimers in a competitive fashion. Although pertuzumab, an antibody binding to the dimerization arm of ErbB2, completely abolished the formation of constitutive and HRG-induced ErbB3/ErbB2 heterodimers, it only slightly blocked ErbB3 homodimerization. The results imply that a dynamic equilibrium exists between constitutive and ligand-induced homo- and heterodimers capable of shaping transmembrane signaling.

EGF Receptor Stalls upon Activation as Evidenced by Complementary Fluorescence Correlation Spectroscopy and Fluorescence Recovery after Photobleaching Measurements.

To elucidate the molecular details of the activation-associated clustering of epidermal growth factor receptors (EGFRs), the time course of the mobility and aggregation states of eGFP tagged EGFR in the membranes of Chinese hamster ovary (CHO) cells was assessed by in situ mobility assays. Fluorescence correlation spectroscopy (FCS) was used to probe molecular movements of small ensembles of molecules over short distances and time scales, and to report on the state of aggregation. The diffusion of larger ensembles of molecules over longer distances (and time scales) was investigated by fluorescence recovery after photobleaching (FRAP). Autocorrelation functions could be best fitted by a two-component diffusion model corrected for triplet formation and blinking. The slow, 100-1000 ms component was attributed to membrane localized receptors moving with free Brownian diffusion, whereas the fast, ms component was assigned to cytosolic receptors or their fragments. Upon stimulation with 50 nM EGF, a significant decrease from 0.11 to 0.07 µm2/s in the diffusion coefficient of membrane-localized receptors was observed, followed by recovery to the original value in ~20 min. In contrast, the apparent brightness of diffusing species remained the same. Stripe FRAP experiments yielded a decrease in long-range molecular mobility directly after stimulation, evidenced by an increase in the recovery time of the slow component from 13 to 21.9 s. Our observations are best explained by the transient attachment of ligand-bound EGFRs to immobile or slowly moving structures such as the cytoskeleton or large, previously photobleached receptor aggregates.

Reducing the Detrimental Effects of Saturation Phenomena in FRET Microscopy.

Although Förster resonance energy transfer (FRET) is one of the most widely used biophysical methods in biology, the effect of high excitation intensity, leading to donor and acceptor saturation, has not been addressed previously. Here, we present a formalism for the experimental determination of the FRET efficiency at high excitation intensity when saturation of both the donor and the acceptor significantly affect conventional FRET calculations. We show that the proposed methodology significantly reduces the dependence of the FRET efficiency on excitation intensity, which otherwise significantly distorts FRET calculations at high excitation intensities commonly used in experiments. The work presented here adds additional rigor to the FRET-based investigation of protein interactions and strengthens the device independence of such results.

Molecular Mechanisms and Bioavailability of Polyphenols in Prostate Cancer.

Prostate cancer is the one of the most frequently diagnosed cancers among men over the age of 50. Several lines of evidence support the observation that polyphenols have preventive and therapeutic effects in prostate cancer. Moreover, prostate cancer is ideal for chemoprevention due to its long latency. We propose here an equilibrated lifestyle with a diet rich in polyphenols as prophylactic attempts to slow down the progression of localized prostate cancer or prevent the occurrence of the disease. In this review, we will first summarize the molecular mechanisms of polyphenols in prostate cancer with a focus on the antioxidant and pro-oxidant effects, androgen receptors (AR), key molecules involved in AR signaling and their transactivation pathways, cell cycle, apoptosis, angiogenesis, metastasis, genetic aspects, and epigenetic mechanisms. The relevance of the molecular mechanisms is discussed in light of current bioavailability data regarding the activity of polyphenols in prostate cancer. We also highlight strategies for improving the bioavailability of polyphenols. We hope that this review will lead to further research regarding the bioavailability and the role of polyphenols in prostate cancer prevention and treatment.

Cancer-derived exosomes from HER2-positive cancer cells carry trastuzumab-emtansine into cancer cells leading to growth inhibition and caspase activation.

BACKGROUND: Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate that carries a cytotoxic drug (DM1) to HER2-positive cancer. The target of T-DM1 (HER2) is present also on cancer-derived exosomes. We hypothesized that exosome-bound T-DM1 may contribute to the activity of T-DM1. METHODS: Exosomes were isolated from the cell culture medium of HER2-positive SKBR-3 and EFM-192A breast cancer cells, HER2-positive SNU-216 gastric cancer cells, and HER2-negative MCF-7 breast cancer cells by serial centrifugations including two ultracentrifugations, and treated with T-DM1. T-DM1 not bound to exosomes was removed using HER2-coated magnetic beads. Exosome samples were analyzed by electron microscopy, flow cytometry and Western blotting. Binding of T-DM1-containing exosomes to cancer cells and T-DM1 internalization were investigated with confocal microscopy. Effects of T-DM1-containg exosomes on cancer cells were investigated with the AlamarBlue cell proliferation assay and the Caspase-Glo 3/7 caspase activation assay. RESULTS: T-DM1 binds to exosomes derived from HER2-positive cancer cells, but not to exosomes derived from HER2-negative MCF-7 cells. HER2-positive SKBR-3 cells accumulated T-DM1 after being treated with T-DM1-containg exosomes, and treatment of SKBR-3 and EFM-192A cells with T-DM1-containing exosomes resulted in growth inhibition and activation of caspases 3 and/or 7. CONCLUSION: T-DM1 binds to exosomes derived from HER2-positive cancer cells, and T-DM1 may be carried to other cancer cells via exosomes leading to reduced viability of the recipient cells. The results suggest a new mechanism of action for T-DM1, mediated by exosomes derived from HER2-positive cancer.

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.

Alterations in the properties of the cell membrane due to glycosphingolipid accumulation in a model of Gaucher disease.

Gaucher disease is a lysosomal storage disease characterized by the malfunction of glucocerebrosidase resulting in the accumulation of glucosylceramide and other sphingolipids in certain cells. Although the disease symptoms are usually attributed to the storage of undigested substrate in lysosomes, here we show that glycosphingolipids accumulating in the plasma membrane cause profound changes in the properties of the membrane. The fluidity of the sphingolipid-enriched membrane decreased accompanied by the enlargement of raft-like ordered membrane domains. The mobility of non-raft proteins and lipids was severely restricted, while raft-resident components were only mildly affected. The rate of endocytosis of transferrin receptor, a non-raft protein, was significantly retarded in Gaucher cells, while the endocytosis of the raft-associated GM1 ganglioside was unaffected. Interferon-γ-induced STAT1 phosphorylation was also significantly inhibited in Gaucher cells. Atomic force microscopy revealed that sphingolipid accumulation was associated with a more compliant membrane capable of producing an increased number of nanotubes. The results imply that glycosphingolipid accumulation in the plasma membrane has significant effects on membrane properties, which may be important in the pathogenesis of Gaucher disease.

Epigallocatechin-3-O-gallate alleviates the malignant phenotype in A-431 epidermoid and SK-BR-3 breast cancer cell lines.

In this study, we evaluated the effects of epigallocatechin-3-O-gallate (EGCG) in two cancer cell lines, A-431 overexpressing ErbB1 and SK-BR-3, overexpressing ErbB2. EGCG treatment showed dose-dependent collapse of mitochondrial membrane potential (Δψm), increase in reactive oxygen species (ROS) production, changes in nuclear morphology and reduced viability. Flow cytometry data indicated that EGCG partially decreases the phosphorylation of several proteins involved in cell proliferation and survival: pErbB1(Y1173, Y1068), pAkt(S473) and pERK(Y204). EGCG affected the clonogenic growth in both cell lines with an EC50 of 2.5 and 5.4 µM for A-431 and SK-BR-3, respectively. Wound scratch assay demonstrated that EGCG inhibited the healing in dose-dependent manner and the effect was correlated with partial reduction in phosphorylation of pFAK(S910). Our data suggest that EGCG administration might reduce the unfavourable traits, particularly associated with ErbB1/EGFR overexpression.

Quantitating ADCC against adherent cells: Impedance-based detection is superior to release, membrane permeability, or caspase activation assays in resolving antibody dose response.

Monoclonal antibody-based immunotherapeutics will dominate Pharma's next generation of blockbuster drugs, and Fc-associated functions, including antibody dependent cellular cytotoxicity (ADCC) are among the highly desired activities mediated by these antibodies. Therefore, quantitative evaluation of ADCC is required during drug development. Our objective was to find the most suitable and reliable nonradioactive method for quantitative analysis of in vitro ADCC against adherent cells, which often serve as models for solid tumors. The test system was comprised the HER2 positive JIMT-1 cells targeted by the specific therapeutic antibodies trastuzumab (Herceptin® ) and pertuzumab (Perjeta® ). These cells are resistant to the direct biological effects of these antibodies, and, therefore, allow the isolated assessment of ADCC. We compared fluorescein diacetate (FDA) and carboxyfluorescein diacetate succinimidyl ester (CFSE) release as a fluorescent alternative to 51 Cr release; propidium iodide (PI) uptake revealing increased membrane permeability; the PanToxiLux assay measuring ADCC induced pro-apoptotic protease activity in flow cytometry; and an impedance-based real time cell adhesion test. We found that release assays are compromised by high spontaneous release of the label. PI uptake could not differentiate well between spontaneous NK activity and specific ADCC. The PanToxiLux assay, besides allowing for shorter assay times, offers improvement over the previous approaches in distinguishing spontaneous and antibody mediated NK action, but, probably owed to the prolonged detached state of adherent target cells, only at highly saturating antibody concentrations. In the case of adherent target cells, impedance-based cell analysis attains functional information exclusively on the target cells without having to label them for distinguishing from effectors or assay readout. It also allows continuous monitoring for days, and specifically detects target cell detachment, as the final functional consequence of ADCC. The sensitivity of this method even allows for quantitating the additivity and saturability of ADCC as a function of antibody concentration. We conclude that impedance-based assays are the most sensitive for quantitatively assessing in vitro ADCC on adherent target cells. © 2017 International Society for Advancement of Cytometry.

The dipole potential correlates with lipid raft markers in the plasma membrane of living cells.

The dipole potential generating an electric field much stronger than any other type of membrane potential influences a wide array of phenomena, ranging from passive permeation to voltage-dependent conformational changes of membrane proteins. It is generated by the ordered orientation of lipid carbonyl and membrane-attached water dipole moments. Theoretical considerations and indirect experimental evidence obtained in model membranes suggest that the dipole potential is larger in liquid-ordered domains believed to correspond to lipid rafts in cell membranes. Using three different dipole potential-sensitive fluorophores and four different labeling approaches of raft and nonraft domains, we showed that the dipole potential is indeed stronger in lipid rafts than in the rest of the membrane. The magnitude of this difference is similar to that observed between the dipole potential in control and sphingolipid-enriched cells characteristic of Gaucher's disease. The results established that the heterogeneity of the dipole potential in living cell membranes is correlated with lipid rafts and imply that alterations in the lipid composition of the cell membrane in human diseases can lead to substantial changes in the dipole potential.

The Dipole Potential Modifies the Clustering and Ligand Binding Affinity of ErbB Proteins and Their Signaling Efficiency.

Although activation of the ErbB family of receptor tyrosine kinases (ErbB1-4) is driven by oligomerization mediated by intermolecular interactions between the extracellular, the kinase and the transmembrane domains, the transmembrane domain has been largely neglected in this regard. The largest contributor to the intramembrane electric field, the dipole potential, alters the conformation of transmembrane peptides, but its effect on ErbB proteins is unknown. Here, we show by Förster resonance energy transfer (FRET) and number and brightness (N&B) experiments that the epidermal growth factor (EGF)-induced increase in the homoassociation of ErbB1 and ErbB2 and their heteroassociation are augmented by increasing the dipole potential. These effects were even more pronounced for ErbB2 harboring an activating Val → Glu mutation in the transmembrane domain (NeuT). The signaling capacity of ErbB1 and ErbB2 was also correlated with the dipole potential. Since the dipole potential decreased the affinity of EGF to ErbB1, the augmented growth factor-induced effects at an elevated dipole potential were actually induced at lower receptor occupancy. We conclude that the dipole potential plays a permissive role in the clustering of ErbB receptors and that the effects of lipid rafts on ligand binding and receptor signaling can be partially attributed to the dipole potential.

The combination of trastuzumab and pertuzumab administered at approved doses may delay development of trastuzumab resistance by additively enhancing antibody-dependent cell-mediated cytotoxicity.

Although the recently concluded CLEOPATRA trial showed clinical benefits of combining trastuzumab and pertuzumab for treating HER2-positive metastatic breast cancer, trastuzumab monotherapy is still the mainstay in adjuvant settings. Since trastuzumab resistance occurs in over half of these cancers, we examined the mechanisms by which treatment of intrinsically trastuzumab-resistant and -sensitive tumors can benefit from the combination of these antibodies. F(ab')2 of both trastuzumab and pertuzumab were generated and validated in order to separately analyze antibody-dependent cell-mediated cytotoxicity (ADCC)-based and direct biological effects of the antibodies. Compared to monotherapy, combination of the two antibodies at clinically permitted doses enhanced the recruitment of natural killer cells responsible for ADCC, and significantly delayed the outgrowth of xenografts from intrinsically trastuzumab-resistant JIMT-1 cells. Antibody dose-response curves of in vitro ADCC showed that antibody-mediated killing can be saturated, and the two antibodies exert an additive effect at sub-saturation doses. Thus, the additive effect in vivo indicates that therapeutic tissue levels likely do not saturate ADCC. Additionally, isobole studies with the in vitro trastuzumab-sensitive BT-474 cells showed that the direct biological effect of combined treatment is additive, and surpasses the maximum effect of either monotherapy. Our results suggest the combined therapy is expected to give results that are superior to monotherapy, whatever the type of HER2-positive tumor may be. The combination of both antibodies at maximum clinically approved doses should thus be administered to patients to recruit maximum ADCC and cause maximum direct biological growth inhibition.

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.