Gerrit Jansen

Inflamed skeletal muscle is challenging for imaging-based spatial transcriptomics because large multinucleated myofibers are poorly represented by nucleus-centred segmentation. We developed a compartment-first Xenium workflow in biopsies from 11 patients with inflammatory muscle disease. StarDist first detected nucleated objects, which were consolidated and subtracted from the tissue region. Cellpose then segmented the residual sarcolemmal myofiber compartment, after which individual nucleated objects were restored, and all geometries were imported into Xenium Ranger for transcript reassignment. Across all samples, the median transcripts per segmented object increased from 33 with the default Xenium segmentation to 68 after custom reassignment and to 75 after quality-control filtering. The workflow improved anatomical representation of myofibers, preserved nucleated interstitial compartments, and supported clustering of expected muscle, immune, vascular, and stromal populations. Open-source scripts provide a reproducible framework for Xenium analysis of inflamed skeletal muscle.

Aim: TNF-related apoptosis-inducing ligand (TRAIL) is a promising targeted anti-cancer agent for several types of cancer, including non-small cell lung cancer (NSCLC). The proteasome inhibitor bortezomib can further potentiate rhTRAIL-induced apoptosis in NSCLC cells. Here, the mechanisms underlying this sensitization were examined in TRAIL-sensitive H460 and TRAIL-resistant A549 and SW1573 NSCLC cells. Methods: NSCLC cell lines were treated with rhTRAIL and bortezomib, and apoptosis was assessed through caspase activation assays, western blotting, and gene silencing of key apoptotic regulators, including Bid, XIAP, and cFLIP. Clonogenic assays were performed to evaluate long-term tumor growth suppression. Results: Bortezomib sensitization mechanisms varied across NSCLC cell lines. Combined rhTRAIL/bortezomib treatment enhanced apoptosis across all cell lines. In TRAIL-sensitive H460 cells, rapid caspase activation was observed, with both extrinsic and intrinsic apoptotic pathways contributing to cell death. Sensitization in H460 cells was predominantly mediated via the caspase-8/Bid amplification loop. In A549 cells, the bortezomib sensitizing effect also relied on the caspase-8/Bid amplification loop. Additionally, the inhibition of Bid and XIAP emphasized the critical role of mitochondrial pathways in apoptosis. In SW1573 cells, limited caspase cleavage was detected, with distinct cleavage patterns suggesting cell-specific apoptotic mechanisms. In this cell line, bortezomib primarily enhanced the extrinsic apoptotic pathway, with XIAP depression further increasing apoptosis. Silencing cFLIP, a caspase-8 inhibitor, significantly improved rhTRAIL sensitivity, emphasizing the critical role of caspase-8 activation in overcoming resistance in SW1573. The clonogenic assay demonstrated that bortezomib combined with rhTRAIL significantly suppressed tumor growth, especially in resistant cell lines. Conclusions: This study underscores bortezomib’s ability to differentially enhance rhTRAIL-induced apoptosis by targeting multiple apoptotic regulators. The variety of effects that bortezomib can exert to enhance rhTRAIL-induced apoptosis makes it a very powerful combination for the treatment of NSCLC and various other types of cancer cells.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation leading to joint damage and systemic complications. Angiogenesis promotes inflammation and contributes to RA progression. This study evaluated potential anti-angiogenic effects of several compounds including small-molecule kinase inhibitors, such as sunitinib (pan-kinase inhibitor), tofacitinib (JAK-inhibitor), NIKi (NF-κB-inducing kinase inhibitor), and the integrin-targeting peptide fluciclatide, using a scratch assay and 3D spheroid-based models of angiogenesis. For all drugs tested in the low micromolar range (1–25 μM), sunitinib (as positive anti-angiogenetic control) showed marked inhibition of endothelial cell (EC) migration and sprouting, effectively reducing both scratch closure and sprout formation. Tofacitinib exhibited marginal effectiveness in the scratch assay, but performed better in the 3D models (55% inhibition), whereas NIKi showed around 50% anti-angiogenic effects in both models. Fluciclatide changed EC morphology rather than migration, and only when stimulated with synovial fluid in spheroid model did it show inhibitory effects (at ≥2.5 µM), with none below this dosage. These results highlight the potential of NIKi and tofacitinib for angiogenesis inhibition and of fluciclatide for safe diagnostic targeting of microdose in RA, as well as the need for advanced screening models that mimic RA’s complex inflammatory pro-angiogenic environment.

Aim: The therapeutic targeting of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors in cancer, including non-small cell lung cancer (NSCLC), is a widely studied approach for tumor selective apoptotic cell death therapy. However, apoptosis resistance is often encountered. The main aim of this study was to investigate the apoptotic mechanism underlying TRAIL sensitivity in three bortezomib (BTZ)-resistant NSCLC variants, combining induction of both the intrinsic and extrinsic pathways. Methods: Sensitivity to TRAIL in BTZ-resistant variants was determined using a tetrazolium (MTT) and a clonogenic assay. A RT-qPCR profiling mRNA array was used to determine apoptosis pathway-specific gene expression. The expression of these proteins was determined through ELISA assays and western Blotting, while apoptosis (sub-G1) and cytokine expression were determined using flow cytometry. Apoptotic genes were silenced by specific siRNAs. Lipid rafts were isolated with fractional ultracentrifugation. Results: A549BTZR (BTZ-resistant) cells were sensitive to TRAIL in contrast to parental A549 cells, which are resistant to TRAIL. TRAIL-sensitive H460 cells remained equally sensitive for TRAIL as H460BTZR. In A549BTZR cells, we identified an increased mRNA expression of TNFRSF11B [osteoprotegerin (OPG)] and caspase-1, -4 and -5 mRNAs involved in cytokine activation and immunogenic cell death. Although the OPG, interleukin-6 (IL-6), and interleukin-8 (IL-8) protein levels were markedly enhanced (122-, 103-, and 11-fold, respectively) in the A549BTZR cells, this was not sufficient to trigger TRAIL-induced apoptosis in the parental A549 cells. Regarding the extrinsic apoptotic pathway, the A549BTZR cells showed TRAIL-R1-dependent TRAIL sensitivity. The shift of TRAIL-R1 from non-lipid into lipid rafts enhanced TRAIL-induced apoptosis. In the intrinsic apoptotic pathway, a strong increase in the mRNA and protein levels of the anti-apoptotic myeloid leukemia cell differentiation protein (Mcl-1) and B-cell leukemia/lymphoma 2 (Bcl-2) was found, whereas the B-cell lymphoma-extra large (Bcl-xL) expression was reduced. However, the stable overexpression of Bcl-xL in the A549BTZR cells did not reverse the TRAIL sensitivity in the A549BTZR cells, but silencing of the BH3 Interacting Domain Death Agonist (BID) protein demonstrated the importance of the intrinsic apoptotic pathway, regardless of Bcl-xL. Conclusion: In summary, increased sensitivity to TRAIL-R1 seems predominantly related to the relocalization into lipid rafts and increased extrinsic and intrinsic apoptotic pathways.

Macrophages constitute important immune cell targets of the antifolate methotrexate (MTX) in autoimmune diseases, including rheumatoid arthritis. Regulation of folate/MTX metabolism remains poorly understood upon pro-inflammatory (M1-type/GM-CSF-polarized) and anti-inflammatory (M2-type/M-CSF-polarized) macrophages. MTX activity strictly relies on the folylpolyglutamate synthetase (FPGS) dependent intracellular conversion and hence retention to MTX-polyglutamate (MTX-PG) forms. Here, we determined FPGS pre-mRNA splicing, FPGS enzyme activity and MTX-polyglutamylation in human monocyte-derived M1- and M2-macrophages exposed to 50 nmol/L MTX ex vivo. Moreover, RNA-sequencing analysis was used to investigate global splicing profiles and differential gene expression in monocytic and MTX-exposed macrophages. Monocytes displayed six–eight-fold higher ratios of alternatively-spliced/wild type FPGS transcripts than M1- and M2-macrophages. These ratios were inversely associated with a six–ten-fold increase in FPGS activity in M1- and M2-macrophages versus monocytes. Total MTX-PG accumulation was four-fold higher in M1- versus M2-macrophages. Differential splicing after MTX-exposure was particularly apparent in M2-macrophages for histone methylation/modification genes. MTX predominantly induced differential gene expression in M1-macrophages, involving folate metabolic pathway genes, signaling pathways, chemokines/cytokines and energy metabolism. Collectively, macrophage polarization-related differences in folate/MTX metabolism and downstream pathways at the level of pre-mRNA splicing and gene expression may account for variable accumulation of MTX-PGs, hence possibly impacting MTX treatment efficacy.

Aim: This study aimed to decipher the molecular mechanism underlying the synergistic effect of inhibitors of the mevalonate-cholesterol pathway (i.e., statins) and aminopeptidase inhibitors (APis) on APi-sensitive and -resistant acute myeloid leukemia (AML) cells. Methods: U937 cells and their sublines with low and high levels of acquired resistance to (6S)-[(R)-2-((S)-Hydroxy-hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3,3 dimethyl-butyric acid cyclopentyl ester (CHR2863), an APi prodrug, served as main AML cell line models. Drug combination effects were assessed with CHR2863 and in vitro non-toxic concentrations of various statins upon cell growth inhibition, cell cycle effects, and apoptosis induction. Mechanistic studies involved analysis of Rheb prenylation required for mTOR activation. Results: A strong synergy of CHR2863 with the statins simvastatin, fluvastatin, lovastatin, and pravastatin was demonstrated in U937 cells and two CHR2863-resistant sublines. This potent synergy between simvastatin and CHR2863 was also observed with a series of other human AML cell lines (e.g., THP1, MV4-11, and KG1), but not with acute lymphocytic leukemia or multiple solid tumor cell lines. This synergistic activity was: (i) specific for APis (e.g., CHR2863 and Bestatin), rather than for other cytotoxic agents; and (ii) corroborated by enhanced induction of apoptosis and cell cycle arrest which increased the sub-G1 fraction. Consistently, statin potentiation of CHR2863 activity was abrogated by co-administration of mevalonate and/or farnesyl pyrophosphate, suggesting the involvement of protein prenylation; this was experimentally confirmed by impaired Rheb prenylation by simvastatin. Conclusion: These novel findings suggest that the combined inhibitory effect of impaired Rheb prenylation and CHR2863-dependent mTOR inhibition instigates a potent synergistic inhibition of statins and APis on human AML cells.

Objective To investigate the pharmacokinetics of methotrexate polyglutamate (MTX-PG) accumulation in red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) in patients with early rheumatoid arthritis (RA) after oral and subcutaneous MTX treatment. Methods In a clinical prospective cohort study (Methotrexate Monitoring study), newly diagnosed patients with RA were randomised for oral or subcutaneous MTX. At 1, 2, 3 and 6 months after therapy initiation, blood was collected and RBCs and PBMCs were isolated. MTX-PG_1-6 concentrations were determined by mass spectrometry methods using stable isotopes of MTX-PG_1-6 as internal standards. Results 43 patients (mean age: 58.5 years, 77% female) were included. PBMCs and RBCs revealed disparate pharmacokinetic profiles in both absolute MTX-PG accumulation levels and distribution profiles. Intracellular MTX-PG accumulation in PBMCs was significantly (pPG₂>PG₃). Remarkably, the distribution profile in PBMCs remained constant over 6 months. RBCs accumulated mainly MTX-PG₁ and lower levels of MTX-PG_2-5 at t=1 month. After 3 months, MTX-PG₃ was the main PG-moiety in RBCs, a profile retained after 6 months of MTX therapy. Subcutaneous MTX administration results in higher RBC drug levels than after oral administration, especially shortly after treatment initiation. Conclusions This is the first study reporting disparate MTX-PG accumulation profiles in RBCs versus PBMCs in newly diagnosed patients with RA during 6 months oral or subcutaneous MTX administration. This analysis can contribute to improved MTX therapeutic drug monitoring for patients with RA. Trial registration number NTR 7149.

Extensive angiogenesis is a characteristic feature in the synovial tissue of rheumatoid arthritis (RA) from a very early stage of the disease onward and constitutes a crucial event for the development of the proliferative synovium. This process is markedly intensified in patients with prolonged disease duration, high disease activity, disease severity, and significant inflammatory cell infiltration. Angiogenesis is therefore an interesting target for the development of new therapeutic approaches as well as disease monitoring strategies in RA. To this end, nuclear imaging modalities represent valuable non-invasive tools that can selectively target molecular markers of angiogenesis and accurately and quantitatively track molecular changes in multiple joints simultaneously. This systematic review summarizes the imaging markers used for single photon emission computed tomography (SPECT) and/or positron emission tomography (PET) approaches, targeting pathways and mediators involved in synovial neo-angiogenesis in RA.

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The study by Safy-Khan, et al in the current issue of The Journal of Rheumatology¹ reports that in a methotrexate (MTX)-based treatment regimen for patients with early arthritis, current smoking was significantly associated with a smaller reduction of Disease Activity Score in 28 joints (DAS28) over time compared to noncurrent smoking. This negative effect of current smoking on DAS28 was dose-dependent: patients who smoked 10–19 cigarettes per day did worse than patients who smoked 1–9 cigarettes per day.