Universal CAR T cells targeted to HER2 with a biotin-trastuzumab soluble linker penetrate spheroids and large tumor xenografts that are inherently resistant to trastuzumab mediated ADCC.

CAR T cell therapies face challenges in combating solid tumors due to their single-target approach, which becomes ineffective if the targeted antigen is absent or lost. Universal CAR T cells (UniCAR Ts) provide a promising solution by utilizing molecular tags (linkers), such as biotin conjugated to monoclonal antibodies, enabling them to target a variety of tumor antigens. Recently, we showed that conventional CAR T cells could penetrate the extracellular matrix (ECM) of ADCC-resistant tumors, which forms a barrier to therapeutic antibodies. This finding led us to investigate whether UniCAR T cells, targeted by soluble antibody-derived linkers, could similarly tackle ADCC-resistant tumors where ECM restricts antibody penetration. We engineered UniCAR T cells by incorporating a biotin-binding monomeric streptavidin 2 (mSA2) domain for targeting HER2 via biotinylated trastuzumab (BT). The activation and cytotoxicity of UniCAR T cells in the presence or absence of BT were evaluated in conventional immunoassays. A 3D spheroid coculture was set up to test the capability of UniCAR Ts to access ECM-masked HER2+ cells. For in vivo analysis, we utilized a HER2+ xenograft model in which intravenously administered UniCAR T cells were supplemented with intraperitoneal BT treatments. In vitro, BT-guided UniCAR T cells showed effective activation and distinct anti-tumor response. Upon target recognition, IFNγ secretion correlated with BT concentration. In the presence of BT, UniCAR T cells effectively penetrated HER2+ spheroids and induced cell death in their core regions. In vivo, upon intravenous administration of UniCAR Ts, circulating BT linkers immediately engaged the mSA2 domain and directed effector cells to the HER2+ tumors. However, these co-treated mice died early, possibly due to the lung infiltration of UniCAR T cells that could recognize both native biotin and HER2. Our results suggest that UniCAR T cells guided with soluble linkers present a viable alternative to conventional CAR T cells, especially for patients resistant to antibody therapy and those with solid tumors exhibiting high antigenic variability. Critical to their success, however, is the choice of an appropriate binding domain for the CAR and the corresponding soluble linker, ensuring both efficacy and safety in therapeutic applications.

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.

Taking molecular pathology to the next level: Whole slide multicolor confocal imaging with the Pannoramic Confocal digital pathology scanner.

The emergence and fast advance of digital pathology allows the acquisition, digital storage, interactive recall and analysis of morphology at the tissue level. When applying immunohistochemistry, it also affords the correlation of morphology with the expression of one or two specific molecule of interest. The rise of fluorescence pathology scanners expands the number of detected molecules based on multiplex labeling. The Pannoramic Confocal (created by 3DHistech, Hungary) is a first-of-the-kind digital pathology scanner that affords not only multiplexed fluorescent detection on top of conventional transmission imaging, but also confocality. We have benchmarked this scanner in terms of stability, precision, light efficiency, linearity and sensitivity. X-Y stability and relocalisation precision were well below resolution limit (≤50 nm). Light throughput in confocal mode was 4-5 times higher than that of a point scanning confocal microscope, yielding similar calculated confocal intensities but with the potential for improving signal to noise ratio or scan speed. Response was linear with R2 ≥ 0.9996. Calibrated measurements showed that using indirect labeling ≥2000 molecules per cell could be well detected and imaged on the cell surface. Both standard-based and statistical post-acquisition flatfield corrections are implemented. We have also measured the point spread function (PSF) of the instrument. The dimensions of the PSF are somewhat larger and less symmetric than of the theoretical PSF of a conventional CLSM, however, the spatial homogeneity of these parameters allows for obtaining a specific system PSF for each optical path and using it for optional on-the-fly deconvolution. In conclusion, the Pannoramic Confocal provides sensitive, quantitative widefield and confocal detection of multiplexed fluorescence signals, with optical sectioning and 3D reconstruction, in addition to brightfield transmission imaging. High speed scanning of large samples, analysis of tissue heterogeneity, and detection of rare events open up new ways for quantitatively analyzing tissue sections, organoid cultures or large numbers of adherent cells.

Opposing Effects of Chelidonine on Tyrosine and Serine Phosphorylation of STAT3 in Human Uveal Melanoma Cells.

STAT3 is a transcription factor that regulates various cellular processes with oncogenic potential, thereby promoting tumorigenesis when activated uncontrolled. STAT3 activation is mediated by its tyrosine phosphorylation, triggering dimerization and nuclear translocation. STAT3 also contains a serine phosphorylation site, with a postulated regulatory role in STAT3 activation and G2/M transition. Interleukin-6, a major activator of STAT3, is present in elevated concentrations in uveal melanomas, suggesting contribution of dysregulated STAT3 activation to their pathogenesis. Here, we studied the impact of chelidonine on STAT3 signaling in human uveal melanoma cells. Chelidonine, an alkaloid isolated from Chelidonium majus, disrupts microtubules, causes mitotic arrest and provokes cell death in numerous tumor cells. According to our flow cytometry and confocal microscopy data, chelidonine abrogated IL-6-induced activation and nuclear translocation, but amplified constitutive serine phosphorylation of STAT3. Both effects were restricted to a fraction of cells only, in an all-or-none fashion. A partial overlap could be observed between the affected subpopulations; however, no direct connection could be proven. This study is the first proof on a cell-by-cell basis for the opposing effects of a microtubule-targeting agent on the two types of STAT3 phosphorylation.

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.

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.

Comparative biocompatibility and antimicrobial studies of sorbic acid derivates.

Nowadays, the sorbates are the third largest group of antimicrobial preservatives in food and pharmaceutical industries, following the parabens and benzoates whose safety is questioned by recent publications. A disadvantage of sorbates is their pH dependence, as their antimicrobial effect is greatly reduced in alkaline environment. The main, widely used sorbate derivatives are sorbic acid and potassium sorbate, no sorbic acid esters are involved in current industrial application. We aimed to test whether the esters of sorbic acid are capable to extend the antimicrobial spectrum of the original molecule while maintaining its advantageous biocompatibility profile. A comparative biocompatibility study of different derivatives (sorbic acid, potassium sorbate, isopropyl sorbate and ethyl sorbate) was carried out. In vitro cell viability assays of MTT (2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide), Neutral Red (3-amino-7-dimethylamino-2-methylphenazine hydrochloride) and flow cytometry with propidium iodide and annexin were performed on Caco-2 cells. In case of in vivo toxicity study, G. mellonella larvae were injected with different concentrations of the test compounds. Time-kill tests were executed on reference strains of C. albicans, E. coli, and S. aureus. According to the MTT-assay, the IC50 values were the following: ethyl sorbate, sorbic acid <0.045% w/w, isopropyl sorbate 0.32% w/w, potassium sorbate >0.75% w/w, while Neutral Red values were >0.75% w/w for the esters and potassium sorbate and 0.66% w/w for sorbic acid. Flow cytometry results indicated the higher cell damage in case of isopropyl sorbate. However, the cytotoxic results of isopropyl sorbate, in vivo toxicity study on G. mellonella larvae did not show significant mortality. It was found, that the antimicrobial properties of isopropyl sorbate were outstanding compared to sorbic acid and potassium sorbate. These results indicate, that the use of sorbate esters can be advantageous, hence, further toxicity studies are needed to prove their safety.

Fluconazole is not inferior than caspofungin, micafungin or amphotericin B in the presence of 50% human serum against Candida albicans and Candida parapsilosis biofilms.

Biofilm formation is a relevant risk factor for mortality in candidemia. Data about serum-based susceptibility testing against Candida biofilms are scant; therefore, the activity of fluconazole, amphotericin B, caspofungin and micafungin was determined against Candida albicans and C. parapsilosis biofilms with or without 50% human serum using XTT-based assays. Serum caused a remarkable adverse effect regarding biofilm structure for both species. Additionally, the ratio of nonviable cells increased for C. parapsilosis biofilms, as confirmed by fluorescent microscopy and flow cytometry. Despite impaired biofilm development, traditionally biofilm-active antifungals, surprisingly, showed decreased activity against C. albicans biofilms in serum at concentrations ranging from 0.5 to 1 mg/l and from 0.015 to 1 mg/l for amphotericin B and echinocandins, respectively (P < .01-.05). However, C. parapsilosis showed higher susceptibility to these antifungals due to reduced biofilm mass and the fungicidal effect of serum at concentrations ranging from 0.015 to 1 mg/l and from 0.015 to 512 mg/l for amphotericin B and echinocandins, respectively (P < .01-.05). Fluconazole exerted better antifungal activity in serum than traditionally biofilm-active antifungals against both examined biofilms. For fluconazole, significant differences were observed in susceptibility between serum-treated and serum-free biofilms at concentrations ranging from 0.015 to 8 mg/l and from 0.03 to 512 mg/l for C. albicans and C. parapsilosis isolates, respectively (P < .01-.05). The high antifungal activity of fluconazole in 50% serum both against C. albicans and C. parapsilosis biofilms supports the utility of fluconazole prophylaxis to reduce the risk of catheter-associated fungal infections.