BACKGROUND: Accelerated partial breast irradiation (APBI) is an alternative breast-conserving therapy approach where radiation is delivered in less time compared to whole breast irradiation (WBI), resulting in improved patient convenience, less toxicity, and cost savings. This prospective randomized study compares the external beam APBI with commonly used moderate hypofractionated WBI in terms of feasibility, safety, tolerance, and cosmetic effects. METHODS: Early breast cancer patients after partial mastectomy were equally randomized into two arms- external APBI and moderate hypofractionated WBI. External beam technique using available technical innovations commonly used in targeted hypofractionated radiotherapy to minimize irradiated volumes was used (cone beam computed tomography navigation to clips in the tumor bed, deep inspiration breath hold technique, volumetric modulated arc therapy dose application, using flattening filter free beams and the six degrees of freedom robotic treatment couch). Cosmetics results and toxicity were evaluated using questionnaires, CTCAE criteria, and photo documentation. RESULTS: The analysis of 84 patients with a median age of 64 years showed significantly fewer acute adverse events in the APBI arm regarding skin reactions, local and general symptoms during a median follow-up of 37 months (range 21-45 months). A significant difference in favor of the APBI arm in grade ≥ 2 late skin toxicity was observed (p = 0.026). Late toxicity in the breast area (deformation, edema, fibrosis, and pain), affecting the quality of life and cosmetic effect, occurred in 61% and 17% of patients in WBI and APBI arms, respectively. The cosmetic effect was more favorable in the APBI arm, especially 6 to 12 months after the radiotherapy. CONCLUSION: External APBI demonstrated better feasibility and less toxicity than the standard regimen in the adjuvant setting for treating early breast cancer patients. The presented study confirmed the level of evidence for establishing the external APBI in daily clinical practice. TRIAL REGISTRATION: NCT06007118.

• Klíčová slova
• APBI, Adjuvant radiotherapy, Early breast cancer, External beam radiotherapy, Surgical bed,
• MeSH
• kojenec MeSH
• kombinovaná terapie MeSH
• kvalita života MeSH
• lidé MeSH
• mastektomie MeSH
• nádory prsu * radioterapie   chirurgie   patologie   MeSH
• předškolní dítě MeSH
• prospektivní studie MeSH
• segmentální mastektomie MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• randomizované kontrolované studie MeSH

BACKGROUND: Desmocollin-1 (DSC1) is a desmosomal transmembrane glycoprotein that maintains cell-to-cell adhesion. DSC1 was previously associated with lymph node metastasis of luminal A breast tumors and was found to increase migration and invasion of MCF7 cells in vitro. Therefore, we focused on DSC1 role in cellular and molecular mechanisms in luminal A breast cancer and its possible therapeutic modulation. METHODS: Western blotting was used to select potential inhibitor decreasing DSC1 protein level in MCF7 cell line. Using atomic force microscopy we evaluated effect of DSC1 overexpression and modulation on cell morphology. The LC-MS/MS analysis of total proteome on Orbitrap Lumos and RNA-Seq analysis of total transcriptome on Illumina NextSeq 500 were performed to study the molecular mechanisms associated with DSC1. Pull-down analysis with LC-MS/MS detection was carried out to uncover DSC1 protein interactome in MCF7 cells. RESULTS: Analysis of DSC1 protein levels in response to selected inhibitors displays significant DSC1 downregulation (p-value ≤ 0.01) in MCF7 cells treated with NF-κB inhibitor parthenolide. Analysis of mechanic cell properties in response to DSC1 overexpression and parthenolide treatment using atomic force microscopy reveals that DSC1 overexpression reduces height of MCF7 cells and conversely, parthenolide decreases cell stiffness of MCF7 cells overexpressing DSC1. The LC-MS/MS total proteome analysis in data-independent acquisition mode shows a strong connection between DSC1 overexpression and increased levels of proteins LACRT and IGFBP5, increased expression of IGFBP5 is confirmed by RNA-Seq. Pathway analysis of proteomics data uncovers enrichment of proliferative MCM_BIOCARTA pathway including CDK2 and MCM2-7 after DSC1 overexpression. Parthenolide decreases expression of LACRT, IGFBP5 and MCM_BIOCARTA pathway specifically in DSC1 overexpressing cells. Pull-down assay identifies DSC1 interactions with cadherin family proteins including DSG2, CDH1, CDH3 and tyrosine kinase receptors HER2 and HER3; parthenolide modulates DSC1-HER3 interaction. CONCLUSIONS: Our systems biology data indicate that DSC1 is connected to mechanisms of cell cycle regulation in luminal A breast cancer cells, and can be effectively modulated by parthenolide.

• Klíčová slova
• Breast cancer, DIA, DSC1, Metastasis, Proteomics, Pull-down,
• MeSH
• chromatografie kapalinová MeSH
• desmokoliny * metabolismus   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• nádory * MeSH
• proteom MeSH
• seskviterpeny farmakologie   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• desmokoliny * MeSH
• DSC1 protein, human MeSH Prohlížeč
• parthenolide MeSH Prohlížeč
• proteom MeSH
• seskviterpeny MeSH

Nucleic acids aptamers often fail to efficiently target some proteins because of the hydrophilic character of the natural nucleotides. Here we present hydrophobic 7-phenylbutyl-7-deaadenine-modified DNA aptamers against the Heat Shock Protein 70 that were selected via PEX and magnetic bead-based SELEX. After 9 rounds of selection, the pool was sequenced and a number of candidates were identified. Following initial screening, two modified aptamers were chemically synthesised in-house and their binding affinity analysed by two methods, bio-layer interferometry and fluorescent-plate-based binding assay. The binding affinities of the modified aptamers were compared with that of their natural counterparts. The resulting modified aptamers bound with higher affinity (low nanomolar range) to the Hsp70 than their natural sequence (>5 µM) and hence have potential for applications and further development towards Hsp70 diagnostics or even therapeutics.

• Publikační typ
• časopisecké články MeSH

Immune checkpoint inhibitors (ICI) are the main therapy currently used in advanced malignant melanoma (MM) and non-small cell lung cancer (NSCLC). Despite the wide variety of uses, the possibility of predicting ICI efficacy in these tumor types is scarce. The aim of our study was to find new predictive biomarkers for ICI treatment. We analyzed, by immunohistochemistry, various cell subsets, including CD3+, CD8+, CD68+, CD20+, and FoxP3+ cells, and molecules such as LAG-3, IDO1, and TGFβ. Comprehensive genomic profiles were analyzed. We evaluated 46 patients with advanced MM (31) and NSCLC (15) treated with ICI monotherapy. When analyzing the malignant melanoma group, shorter median progression-free survival (PFS) was found in tumors positive for nuclear FoxP3 in tumor-infiltrating lymphocytes (TILs) (p = 0.048, HR 3.04) and for CD68 expression (p = 0.034, HR 3.2). Longer PFS was achieved in patients with tumors with PD-L1 TPS ≥ 1 (p = 0.005, HR 0.26). In the NSCLC group, only FoxP3 positivity was associated with shorter PFS and OS. We found that FoxP3 negativity was linked with a better response to ICI in both histological groups.

• Klíčová slova
• NSCLC, anti-tumor immunity, immune checkpoint inhibitors, malignant melanoma, predictive biomarker,
• Publikační typ
• časopisecké články MeSH

Catechol-O-methyl transferase (COMT) is involved in detoxification of catechol estrogens, playing cancer-protective role in cells producing or utilizing estrogen. Moreover, COMT suppressed migration potential of breast cancer (BC) cells. To delineate COMT role in metastasis of estrogen receptor (ER) dependent BC, we investigated the effect of COMT overexpression on invasion, transcriptome, proteome and interactome of MCF7 cells, a luminal A BC model, stably transduced with lentiviral vector carrying COMT gene (MCF7-COMT). 2D and 3D assays revealed that COMT overexpression associates with decreased cell invasion (p < 0.0001 for Transwell assay, p < 0.05 for spheroid formation). RNA-Seq and LC-DIA-MS/MS proteomics identified genes associated with invasion (FTO, PIR, TACSTD2, ANXA3, KRT80, S100P, PREX1, CLEC3A, LCP1) being downregulated in MCF7-COMT cells, while genes associated with less aggressive phenotype (RBPMS, ROBO2, SELENBP, EPB41L2) were upregulated both at transcript (|log2FC|> 1, adj. p < 0.05) and protein (|log2FC|> 0.58, q < 0.05) levels. Importantly, proteins driving MET signaling were less abundant in COMT overexpressing cells, and pull-down confirmed interaction between COMT and Kunitz-type protease inhibitor 2 (SPINT2), a negative regulator of MET (log2FC = 5.10, q = 1.04-7). In conclusion, COMT may act as tumor suppressor in ER dependent BC not only by detoxification of catechol estrogens but also by suppressing cell invasion and interplay with MET pathway.

• MeSH
• estrogeny metabolismus   MeSH
• katechol-O-methyltransferasa * genetika   MeSH
• katecholestrogeny MeSH
• katecholy MeSH
• nádory * MeSH
• receptory pro estrogeny metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• catechol MeSH Prohlížeč
• estrogeny MeSH
• katechol-O-methyltransferasa * MeSH
• katecholestrogeny MeSH
• katecholy MeSH
• receptory pro estrogeny MeSH

Defining dynamic protein-protein interactions in the ubiquitin conjugation reaction is a challenging research area. Generating peptide aptamers that target components such as ubiquitin itself, E1, E2, or E3 could provide tools to dissect novel features of the enzymatic cascade. Next-generation deep sequencing platforms were used to identify peptide sequences isolated from phage-peptide libraries screened against Ubiquitin and its ortholog NEDD8. In over three rounds of selection under differing wash criteria, over 13,000 peptides were acquired targeting ubiquitin, while over 10,000 peptides were selected against NEDD8. The overlap in peptides against these two proteins was less than 5% suggesting a high degree in specificity of Ubiquitin or NEDD8 toward linear peptide motifs. Two of these ubiquitin-binding peptides were identified that inhibit both E3 ubiquitin ligases MDM2 and CHIP. NMR analysis highlighted distinct modes of binding of the two different peptide aptamers. These data highlight the utility of using next-generation sequencing of combinatorial phage-peptide libraries to isolate peptide aptamers toward a protein target that can be used as a chemical tool in a complex multi-enzyme reaction.

• Klíčová slova
• aptamers, molecular dynamics, next-generation sequencing, phage-peptide, protein–peptide binding, ubiquitin,
• Publikační typ
• časopisecké články MeSH

The in vivo rituximab effects in B cell malignancies are only partially understood. Here we analyzed in a large chronic lymphocytic leukemia (CLL) cohort (n = 80) the inter-patient variability in CLL cell count reduction within the first 24 h of rituximab administration in vivo, and a phenomenon of blood repopulation by malignant cells after anti-CD20 antibody therapy. Larger CLL cell elimination after rituximab infusion was associated with lower pre-therapy CLL cell counts, higher CD20 levels, and the non-exhausted capacity of complement-dependent cytotoxicity (CDC). The absolute amount of cell-surface CD20 molecules (CD20 density x CLL lymphocytosis) was a predictor for complement exhaustion during therapy. We also describe that a highly variable decrease in CLL cell counts at 5 h (88 %-2%) following rituximab infusion is accompanied in most patients by peripheral blood repopulation with CLL cells at 24 h, and in ∼20 % of patients, this resulted in CLL counts higher than before therapy. We provide evidence that CLL cells recrudescence is linked with i) CDC exhaustion, which leads to the formation of an insufficient amount of membrane attack complexes, likely resulting in temporary retention of surviving rituximab-opsonized cells by the mononuclear-phagocyte system (followed by their release back to blood), and ii) CLL cells regression from immune niches (CXCR4dimCD5bright intraclonal subpopulation). Patients with major peripheral blood CLL cell repopulation exhibited a longer time-to-progression after chemoimmunotherapy compared to patients with lower or no repopulation, suggesting chemotherapy vulnerability of CLL cells that repopulate the blood.

• Klíčová slova
• Chronic lymphocytic leukemia/CLL, Complement, Peripheral blood repopulation, Recrudescence, Rituximab,
• MeSH
• chronická lymfatická leukemie krev   farmakoterapie   imunologie   patologie   MeSH
• cytotoxicita imunologická imunologie   MeSH
• komplement imunologie   MeSH
• lidé MeSH
• následné studie MeSH
• protinádorové látky imunologicky aktivní terapeutické užití   MeSH
• rituximab terapeutické užití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• komplement MeSH
• protinádorové látky imunologicky aktivní MeSH
• rituximab MeSH

The detection of cancer antigens is a major aim of cancer research in order to develop better patient management through early disease detection. Many cancers including prostate, lung, and ovarian secrete a protein disulfide isomerase protein named AGR2 that has been previously detected in urine and plasma using mass spectrometry. Here we determine whether a previously developed monoclonal antibody targeting AGR2 can be adapted from an indirect two-site ELISA format into a direct detector using solid-phase printed gold electrodes. The screen-printed gold electrode was surface functionalized with the anti-AGR2 specific monoclonal antibody. The interaction of the recombinant AGR2 protein and the anti-AGR2 monoclonal antibody functionalized electrode changed its electrochemical impedance spectra. Nyquist diagrams were obtained after incubation in an increasing concentration of purified AGR2 protein with a range of concentrations from 0.01 fg/mL to 10 fg/mL. In addition, detection of the AGR2 antigen can be achieved from cell lysates in medium or artificial buffer. These data highlight the utility of an AGR2-specific monoclonal antibody that can be functionalized onto a gold printed electrode for a one-step capture and quantitation of the target antigen. These platforms have the potential for supporting methodologies using more complex bodily fluids including plasma and urine for improved cancer diagnostics.

• Klíčová slova
• AGR2 protein, electrochemical impedance spectroscopy, screen-printed gold electrode, sensor,
• MeSH
• biosenzitivní techniky * MeSH
• elektrochemické techniky MeSH
• elektrody MeSH
• kovové nanočástice MeSH
• lidé MeSH
• limita detekce MeSH
• monoklonální protilátky MeSH
• mukoproteiny analýza   MeSH
• nádory MeSH
• onkogenní proteiny analýza   MeSH
• zlato MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• AGR2 protein, human MeSH Prohlížeč
• monoklonální protilátky MeSH
• mukoproteiny MeSH
• onkogenní proteiny MeSH
• zlato MeSH

Despite proteotoxic stress and heat shock being implicated in diverse pathologies, currently no methodology to inflict defined, subcellular thermal damage exists. Here, we present such a single-cell method compatible with laser-scanning microscopes, adopting the plasmon resonance principle. Dose-defined heat causes protein damage in subcellular compartments, rapid heat-shock chaperone recruitment, and ensuing engagement of the ubiquitin-proteasome system, providing unprecedented insights into the spatiotemporal response to thermal damage relevant for degenerative diseases, with broad applicability in biomedicine. Using this versatile method, we discover that HSP70 chaperone and its interactors are recruited to sites of thermally damaged proteins within seconds, and we report here mechanistically important determinants of such HSP70 recruitment. Finally, we demonstrate a so-far unsuspected involvement of p97(VCP) translocase in the processing of heat-damaged proteins. Overall, we report an approach to inflict targeted thermal protein damage and its application to elucidate cellular stress-response pathways that are emerging as promising therapeutic targets.

• MeSH
• analýza jednotlivých buněk metody   MeSH
• kovové nanočástice chemie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• povrchová plasmonová rezonance MeSH
• proteasomový endopeptidasový komplex metabolismus   MeSH
• protein obsahující valosin genetika   metabolismus   MeSH
• proteiny tepelného šoku HSP70 metabolismus   MeSH
• reakce na tepelný šok * MeSH
• stříbro chemie   MeSH
• ubikvitin metabolismus   MeSH
• vysoká teplota škodlivé účinky   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteasomový endopeptidasový komplex MeSH
• protein obsahující valosin MeSH
• proteiny tepelného šoku HSP70 MeSH
• stříbro MeSH
• ubikvitin MeSH
• VCP protein, human MeSH Prohlížeč

A comparative canine-human therapeutics model is being developed in B-cell lymphoma through the generation of a hybridoma cell that produces a murine monoclonal antibody specific for canine CD20. The hybridoma cell produces two light chains, light chain-3, and light chain-7. However, the contribution of either light chain to the authentic full-length hybridoma derived IgG is undefined. Mass spectrometry was used to identify only one of the two light chains, light chain-7, as predominating in the full-length IgG. Gene synthesis created a recombinant murine-canine chimeric monoclonal antibody expressing light chain-7 that reconstituted the IgG binding to CD20. Using light chain-7 as a reference sequence, hydrogen deuterium exchange mass spectrometry was used to identify the dominant CDR region implicated in CD20 antigen binding. Early in the deuteration reaction, the CD20 antigen suppressed deuteration at CDR3 (VH). In later time points, deuterium suppression occurred at CDR2 (VH) and CDR2 (VL), with the maintenance of the CDR3 (VH) interaction. These data suggest that CDR3 (VH) functions as the dominant antigen docking motif and that antibody aggregation is induced at later time points after antigen binding. These approaches define a methodology for fine mapping of CDR contacts using nested enzymatic reactions and hydrogen deuterium exchange mass spectrometry. These data support the further development of an engineered, synthetic canine-murine monoclonal antibody, focused on CDR3 (VH), for use as a canine lymphoma therapeutic that mimics the human-murine chimeric anti-CD20 antibody Rituximab.

• Klíčová slova
• CD20, comparative medicine, hydrogen deuterium exchange mass spectrometry, lymphoma, monoclonal antibody,
• MeSH
• antigeny CD20 imunologie   MeSH
• chromatografie kapalinová MeSH
• imunoglobulin G chemie   MeSH
• kinetika MeSH
• lehké řetězce imunoglobulinů genetika   metabolismus   MeSH
• lidé MeSH
• monoklonální protilátky chemie   genetika   MeSH
• nádorové buněčné linie MeSH
• peptidová knihovna MeSH
• psi MeSH
• rekombinantní fúzní proteiny MeSH
• sekvence aminokyselin MeSH
• tandemová hmotnostní spektrometrie MeSH
• těžké řetězce imunoglobulinů genetika   metabolismus   MeSH
• vazebná místa protilátek MeSH
• vodík/deuteriová výměna a hmotnostní spektrometrie * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD20 MeSH
• imunoglobulin G MeSH
• lehké řetězce imunoglobulinů MeSH
• monoklonální protilátky MeSH
• peptidová knihovna MeSH
• rekombinantní fúzní proteiny MeSH
• těžké řetězce imunoglobulinů MeSH