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.

Novel Transmission Electron Microscopic Method to Quantify Protein Clustering on the Cell Surface.

The cell surface distribution patterns (clustering) of membrane proteins have been widely investigated in cell biology. Here we describe a novel transmission electron microscopic (TEM) protocol designed to improve the quality of information obtained about the protein distribution patterns detected. This novel method makes it possible to study the clustering of all transmembrane proteins on one half of the cytoplasmic membrane of a whole cell. To achieve better imaging, we combine various methods, including critical-point drying, fixation of gold beads with a carbon layer, and a newly developed chemical thinning method. In addition, in our image-processing algorithm, we implemented pair correlation and pair cross-correlation functions, providing more details and better quantitative accuracy in characterizing the size and numbers of possible protein clusters. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation and transmission electron micrography Alternate Protocol: Direct cell labeling for transmission electron micrography Basic Protocol 2: Analysis of TEM images to detect immunogold-labeled proteins.

HLA DQ protein changes the cell surface distribution pattern of HLA proteins as monitored by Förster resonance energy transfer and high-resolution electron microscopy.

Peptide presentation by MHC class I and MHC class II molecules plays important roles in the regulation of the immune response. One factor in these displays is the density of antigen, which must exceed a critical threshold for the effective activation of T cells. Nonrandom distribution of MHC class I and class II has already been detected at the nanometer level and at higher hierarchical levels. It is not clear how the absence and reappearance of some protein molecules can influence the nonrandom distribution. Therefore, we performed experiments on HLA II-deficient bare lymphocyte syndrome (BLS1) cells: we created a stable transfected cell line, tDQ6-BLS-1, and were able to detect the effect of the appearance of HLA-DQ6 molecules on the homo and heteroassociation of different cell surface molecules by comparing Förster resonance energy transfer (FRET) efficiency on transfected cells to that on nontransfected BLS-1 and JY human B-cell lines. Our FRET results show a decrease in homoassociation FRET between HLA I chains in HLA-DQ6-transfected tDQ6-BLS-1 cells compared with the parent BLS-1 cell line and an increase in heteroassociation FRET between HLA I and HLA II (compared with JY cells), suggesting a similar pattern of antigen presentation by the HLA-DQ6 allele. Transmission electron microscopy (TEM) revealed that both HLA class I and class II molecules formed clusters at higher hierarchical levels on the tDQ6-BLS-1 cells, and the de novo synthesized HLA DQ molecules did not intersperse with HLA class I islands. These observations could be important in understanding the fine tuning of the immune response.

Pixel-by-pixel autofluorescence corrected FRET in fluorescence microscopy improves accuracy for samples with spatially varied autofluorescence to signal ratio.

The actual interaction between signaling species in cellular processes is often more important than their expression levels. Förster resonance energy transfer (FRET) is a popular tool for studying molecular interactions, since it is highly sensitive to proximity in the range of 2-10 nm. Spectral spillover-corrected quantitative (3-cube) FRET is a cost effective and versatile approach, which can be applied in flow cytometry and various modalities of fluorescence microscopy, but may be hampered by varying levels of autofluorescence. Here, we have implemented pixel-by-pixel autofluorescence correction in microscopy FRET measurements, exploiting cell-free calibration standards void of autofluorescence that allow the correct determination of all spectral spillover factors. We also present an ImageJ/Fiji plugin for interactive analysis of single images as well as automatic creation of quantitative FRET efficiency maps from large image sets. For validation, we used bead and cell based FRET models covering a range of signal to autofluorescence ratios and FRET efficiencies and compared the approach with conventional average autofluorescence/background correction. Pixel-by-pixel autofluorescence correction proved to be superior in the accuracy of results, particularly for samples with spatially varying autofluorescence and low fluorescence to autofluorescence ratios, the latter often being the case for physiological expression levels.

Principles of Resonance Energy Transfer.

This unit describes the basic principles of Förster resonance energy transfer (FRET). Beginning with a brief summary of the history of FRET applications, the theory of FRET is introduced in detail using figures to explain all the important parameters of the FRET process. After listing various approaches for measuring FRET efficiency, several pieces of advice are given on choosing the appropriate instrumentation. The unit concludes with a discussion of the limitations of FRET measurements followed by a few examples of the latest FRET applications, including new developments such as spectral flow cytometric FRET, single-molecule FRET, and combinations of FRET with super-resolution or lifetime imaging microscopy and with molecular dynamics simulations. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.

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.

Comprehensive Model for Epidermal Growth Factor Receptor Ligand Binding Involving Conformational States of the Extracellular and the Kinase Domains.

The epidermal growth factor (EGF) receptor (EGFR) undergoes ligand-dependent dimerization to initiate transmembrane signaling. Although crystallographic structures of the extracellular and kinase domains are available, ligand binding has not been quantitatively analyzed taking the influence of both domains into account. Here, we developed a model explicitly accounting for conformational changes of the kinase and extracellular domains, their dimerizations and ligand binding to monomeric and dimeric receptor species. The model was fitted to ligand binding data of suspended cells expressing receptors with active or inactive kinase conformations. Receptor dimers with inactive, symmetric configuration of the kinase domains exhibit positive cooperativity and very weak binding affinity for the first ligand, whereas dimers with active, asymmetric kinase dimers are characterized by negative cooperativity and subnanomolar binding affinity for the first ligand. The homodimerization propensity of EGFR monomers with active kinase domains is ∼100-times higher than that of dimers with inactive kinase domains. Despite this fact, constitutive, ligand-independent dimers are mainly generated from monomers with inactive kinase domains due to the excess of such monomers in the membrane. The experimental finding of increased positive cooperativity at high expression levels of EGFR was recapitulated by the model. Quantitative prediction of ligand binding to different receptor species revealed that EGF binds to receptor monomers and dimers in an expression-level dependent manner without significant recruitment of monomers to dimers upon EGF stimulation below the phase transition temperature of the membrane. Results of the fitting offer unique insight into the workings of the EGFR.

Quo vadis FRET? Förster's method in the era of superresolution.

Although the theoretical foundations of Förster resonance energy transfer (FRET) were laid in the 1940s as part of the quantum physical revolution of the 20th century, it was only in the 1970s that it made its way to biology as a result of the availability of suitable measuring and labeling technologies. Thanks to its ease of application, FRET became widely used for studying molecular associations on the nanometer scale. The development of superresolution techniques at the turn of the millennium promised an unprecedented insight into the structure and function of molecular complexes. Without downplaying the significance of superresolution microscopies this review expresses our view that FRET is still a legitimate tool in the armamentarium of biologists for studying molecular associations since it offers distinct advantages and overcomes certain limitations of superresolution approaches.

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.