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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.

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.

Background and Objectives: Multiple myeloma (MM) remains an incurable plasma cell malignancy with heterogeneous clinical outcomes. Although current prognostic systems integrate biochemical and cytogenetic parameters, they do not fully capture disease complexity. Adipocytes within the bone marrow microenvironment secrete adipokines that regulate inflammation, metabolism, and immune interactions and may influence disease progression. This study aimed to assess circulating adipokines and related microenvironmental mediators as potential biomarkers of disease activity and treatment response in MM. Materials and Methods: In this case–control, cross-sectional study, the serum levels of eight adipokine-related molecules—adiponectin, leptin, resistin, chemerin, adipsin, thrombospondin-1 (TSP-1), paraoxonase-1 (PON-1), and myeloperoxidase (MPO)—were measured in 40 MM patients and 38 age- and sex-matched healthy controls. Enzyme-linked immunosorbent assays (ELISA) and bead-based multiplex immunoassays were used. Associations with prognostic markers (serum β2-microglobulin (sB2M), LDH, albumin, hemoglobin, renal function) and treatment response were analyzed using correlation and non-parametric statistical methods. Results: Compared to the controls, MM patients exhibited significantly higher circulating levels of adiponectin, resistin, chemerin, adipsin, TSP-1, and MPO, while leptin was decreased. Among clinical correlations, chemerin and PON-1 correlated positively with sB2M, TSP-1 correlated with LDH, and MPO correlated with M-protein and albumin. Resistin was lower in patients with renal impairment and an advanced disease stage. Adiponectin and TSP-1 were significantly lower in progressive disease compared to complete remission, suggesting their potential association with treatment response. Conclusions: This study demonstrates that multiple adipokines are dysregulated in MM and exhibit distinct associations with disease burden, renal function, and therapeutic response. Novel associations identified for TSP-1, PON-1, and adipsin highlight previously unrecognized microenvironmental pathways in MM biology. Adipokine profiling may complement established prognostic markers and provide new insights into the tumour microenvironment in MM.

The interaction between the nervous and immune systems is crucial for maintaining homeostasis and can influence disease progression in inflammatory skin diseases, such as atopic dermatitis (AD). Sensory neurons in the skin can secrete neuropeptides that modulate immune cell activity, including Langerhans cells (LCs), one of the primary antigen-presenting cells in the epidermis. In our study we investigated the effects of neuropeptides on the differentiation of monocyte-derived LCs (moLCs), specifically the neuropeptides with the most profound effect, i.e. atrial- and B-type natriuretic peptides (ANP and BNP, respectively). RNA sequencing and RT-qPCR were used to analyze neuropeptide receptor expression in moLCs and immature dendritic cells (iDCs), and the most translationally relevant, natriuretic peptide receptor A (NPR1) was validated on the protein level using western blotting. Cell surface markers of moLCs were assessed using flow cytometry, and NPR1 functionality was confirmed through intracellular cGMP assays. Confocal microscopy was used to confirm the expression of NPR1 in healthy and AD skin. RNA-Seq analysis was also employed to characterize the phenotypic changes in moLCs differentiated in the presence of BNP. NPR1 expression was significantly higher in moLCs compared to iDCs, and treatment with ANP and BNP enhanced moLC differentiation, increasing CD207, CD1a, and HLA-DQ expression, while other tested neuropeptides (calcitonin gene-related peptide [CGRP], neurotensin) had no significant effect. NPR1 was functionally active, as evidenced by increased intracellular cGMP levels upon ligand binding. Confocal microscopy revealed NPR1 expression on LC cell bodies in both healthy and AD skin, with reduced intensity in AD. RNA-Seq analysis of BNP-treated moLCs indicated a shift toward a migratory LC phenotype, marked by upregulation of genes associated with cell motility (e.g., CCR7, LAMP3). These findings demonstrate that NPR1 activation promotes a migratory LC phenotype, highlighting the role of neuropeptides in shaping cutaneous immune responses. The reduced number of LCs in AD skin suggests a potential link between neuropeptide signaling and disease pathology.

Background/Objectives: Monoclonal antibody therapies for HER2-positive tumors frequently encounter resistance, requiring alternative treatment strategies. This study investigates the use of natural killer (NK) cells expressing HER2-specific chimeric antigen receptor (CAR) to address this issue. CAR NK cells have several benefits over CAR T cells: they are less likely to cause severe side effects such as cytokine release syndrome and neurotoxicity, can be sourced from various origins, and do not trigger Graft versus Host Disease, making them ideal for “off-the-shelf” applications. Methods: We have generated NK-92 cell lines expressing first, second and third-generation HER2-specific CARs with CD28 and/or 41BB costimulatory domains using a retroviral transduction system, followed by FACS sorting and expansion to obtain pure HER2-CAR NK-92 cell products for functional benchmarking. Results: In vitro tests showed that these CAR NK cells were effective against both trastuzumab-sensitive (CD44−) and -resistant (CD44+) tumors in monolayer cultures. However, in three-dimensional spheroid models and in vivo xenografts, they were less effective against CD44+ trastuzumab-resistant tumors. Conclusions: This reduced efficacy highlights the significant role of the tumor microenvironment, particularly the extracellular matrix, in hindering the therapeutic potential of CAR NK cells. Despite the promising in vitro performance of CAR NK cells, this study emphasizes the need for improved strategies to enhance their penetration and effectiveness in resistant tumors: optimizing CAR constructs and devising methods to overcome extracellular matrix barriers are crucial for advancing CAR NK cell therapies in oncology.

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.

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.

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.

A digitális patológia gyors fejlődése lehetővé teszi szöveti szintű morfológiai felvételek készítését. Immunhisztokémiai jelölések alkalmazásával ezek a morfológiai jegyek egy vagy két molekula expressziós szintjével korreláltathatóak. Napjainkban a fluoreszcenciás patológiai szkennerek megjelenése és elterjedése zajlik, melyek lehetővé teszik több molekula azonos mintában történő, multiplex jelölésen alapúló vizsgálatát. A Pannoramic Confocal az első digitális patológiai szkenner, amely a hagyományos transzmissziós és fluoreszcenciás képalkotáson túl konfokalitást is biztosít. A szkennert a konfokális képalkotás, stabilitás, pontosság, linearitás és érzékenység szempontjából vizsgáltuk. A konfokális kép pontkiterjedési függvényét aszimmetrikusnak, de térben invariánsnak mértük; ennek alapján dekonvolúciós algoritmust implementáltunk a műszerhez. Az X-Y dimenzióban mért stabilitás és relokalizációs pontatlanság jóval a felbontási határ alatt volt. Az intenzitásválasz lineáris volt (R² ≥0,9996). A kalibrált mérések azt mutatták, hogy indirekt jelölés alkalmazásával sejtenként ≥ 2000 molekula jól detektálható és leképezhető. A megvilágítást vizsgálva azt találtuk, hogy a mikroszkóp megvilágítása aszimmetrikus és inhomogén. Ennek kiküszöbölésére bevezettünk egy korrekciós módszert, amely alkalmas kisebb területről és térben változatos mintákról készült felvételek megfelelő korrekciójára is, valamint megbízható kvantitatív eredményeket ad. Ennek alkalmazásával, ill. a mérési pontosság ismeretében a Pannoramic Confocal alkalmas lehet szövettani metszeteken molekuláris interakciók mérésére. A Förster rezonancia energiatranszfer (FRET) népszerű eszköz a molekuláris kölcsönhatások tanulmányozására, mivel a 1-10 nm-es tartományban igen érzékeny a távolságra. Az intenzitás alapú, spektrálisan korrigált FRET mérési módszer előnye, hogy háromdimenziós, valamint időfüggő mérésekre is alkalmas. Annak érdekében, hogy használható legyen magas autofluoreszcenciájú metszetek mikroszkópos mérése során pixelenkénti autofluoreszcencia korrekcióval, az áramlási citometriában használt sejtenkénti korrekciós algoritmust adaptáltuk. A kidolgozott pixelenkénti autofluoreszcencia korrekció javítja a mérések pontosságát és különösen alacsony szignál/autofluoreszcencia értékek, valamint térben változatos autofluoreszcenciájú minták esetében ajánlható. Az analízis elvégzésére írt Fiji/ImageJ plugin szabadon elérhető.A mérési eljárást a Pannoramic Confocal készülékkel is teszteltük, ahol a sejteken és fagyasztva metszett szöveten mért FRET hatásfokok a kontrollként használt LSM 880 lézerpásztázó mikroszkóppal mértekkel egyezést mutattak. Ez alapján a pixelenkénti autofluoreszcencia korrigált FRET mérés implementálásra került a Pannoramic Confocal digitális patológiai szkenner szoftverében, megteremtve a diagnosztikai célú FRET mérések metodológiai és instrumentális alapját.