Bio-nano interactions have been extensively explored in nanomedicine to develop selective delivery strategies and reduce systemic toxicity. To enhance the delivery of nanocarriers to cancer cells and improve the therapeutic efficiency, different nanomaterials have been developed. However, the limited clinical translation of nanoparticle-based therapies, largely due to issues associated with poor targeting, requires a deeper understanding of the biological phenomena underlying cell-nanoparticle interactions. In this context, we investigate the molecular and cellular mechanobiology parameters that control such interactions. We demonstrate that the pharmacological inhibition or the genetic ablation of the key mechanosensitive component of the Hippo pathway, i.e., yes-associated protein, enhances nanoparticle internalization by 1.5-fold. Importantly, this phenomenon occurs independently of nanoparticle properties, such as size, or cell properties such as surface area and stiffness. Our study reveals that the internalization of nanoparticles in target cells can be controlled by modulating cell mechanosensing pathways, potentially enhancing nanotherapy specificity.
- MeSH
- adaptorové proteiny signální transdukční metabolismus MeSH
- buněčný převod mechanických signálů účinky léků MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nanočástice * chemie MeSH
- nanomedicína MeSH
- signální dráha Hippo MeSH
- signální proteiny YAP MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The CYCS gene is highly evolutionarily conserved, with only a few pathogenic variants that cause thrombocytopenia-4 (THC4). Here, we report a novel CYCS variant NM_018947.6: c.59C>T [NP_061820.1:p.(Thr20Ile)] segregating with thrombocytopenia in three generations of a Czech family. The phenotype of the patients corresponds to THC4 with platelets of normal size and morphology and dominant inheritance. Intriguingly, a gradual decline in platelet counts was observed across generations. CRISPR/Cas9-mediated gene editing was used to introduce the new CYCS gene variant into a megakaryoblast cell line (MEG-01). Subsequently, the adhesion, shape, size, ploidy, viability, mitochondrial respiration, cytochrome c protein (CYCS) expression, cell surface antigen expression and caspase activity were analysed in cells carrying the studied variant. Interestingly, the variant decreases the expression of CYCS while increasing mitochondrial respiration and the expression of CD9 cell surface antigen. Surprisingly, the variant abates caspase activation, contrasting with previously known effects of other CYCS variants. Some reports indicate that caspases may be involved in thrombopoiesis; thus, the observed dysregulation of caspase activity might contribute to thrombocytopenia. The findings significantly enhance our understanding of the molecular mechanisms underlying inherited thrombocytopenia and may have implications for diagnosis, prognosis and future targeted therapies.
- MeSH
- kaspasy * metabolismus genetika MeSH
- lidé MeSH
- rodokmen MeSH
- trombocytopenie * genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.
- MeSH
- buňky stromatu * metabolismus patologie MeSH
- chronická lymfatická leukemie * patologie genetika farmakoterapie MeSH
- inhibitory proteinkinas farmakologie MeSH
- interleukiny genetika metabolismus MeSH
- kokultivační techniky * MeSH
- lidé MeSH
- ligand CD40 * metabolismus genetika MeSH
- myši MeSH
- proliferace buněk * MeSH
- T-lymfocyty imunologie metabolismus MeSH
- xenogenní modely - testy protinádorové aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
I-Motifs (iM) are non-canonical DNA structures potentially forming in the accessible, single-stranded, cytosine-rich genomic regions with regulatory roles. Chromatin, protein interactions, and intracellular properties seem to govern iM formation at sites with i-motif formation propensity (iMFPS) in human cells, yet their specific contributions remain unclear. Using in-cell NMR with oligonucleotide iMFPS models, we monitor iM-associated structural equilibria in asynchronous and cell cycle-synchronized HeLa cells at 37 °C. Our findings show that iMFPS displaying pHT < 7 under reference in vitro conditions occur predominantly in unfolded states in cells, while those with pHT > 7 appear as a mix of folded and unfolded states depending on the cell cycle phase. Comparing these results with previous data obtained using an iM-specific antibody (iMab) reveals that cell cycle-dependent iM formation has a dual origin, and iM formation concerns only a tiny fraction (possibly 1%) of genomic sites with iM formation propensity. We propose a comprehensive model aligning observations from iMab and in-cell NMR and enabling the identification of iMFPS capable of adopting iM structures under physiological conditions in living human cells. Our results suggest that many iMFPS may have biological roles linked to their unfolded states.
- MeSH
- azidy * MeSH
- benzazepiny * MeSH
- DNA MeSH
- HeLa buňky MeSH
- lidé MeSH
- magnetická rezonanční tomografie * MeSH
- protilátky MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
OBJECTIVE: The pathophysiological processes leading to epileptogenesis and pharmacoresistance in epilepsy have been the subject of extensive preclinical and clinical research. The main impact on clinical practice is the development of new targeted therapies for epilepsy. We studied the importance of neuroinflammation in the development of epileptogenesis and pharmacoresistance in childhood epilepsy patients. METHODS: A cross-sectional study conducted at two epilepsy centers in the Czech Republic compared 22 pharmacoresistant patients and 4 pharmacodependent patients to 9 controls. We analyzed the ProcartaPlexTM 9-Plex immunoassay panel consisting of interleukin (IL)-6, IL-8, IL-10, IL-18, CXCL10/IP-10, monocyte chemoattractant protein 1 (CCL2/MCP-1), B lymphocyte chemoattractant (BLC), tumor necrosis factor-alpha (TNF-α), and chemokine (C-X3-X motif) ligand 1 (fractalkine/CXC3CL1) to determine their alterations in cerebrospinal fluid (CSF) and blood plasma, concurrently. RESULTS: The analysis of 21 paired CSF and plasma samples in pharmacoresistant patients compared to controls revealed a significant elevation of CCL2/MCP-1 in CSF (p < 0.000512) and plasma (p < 0.00.017). Higher levels of fractalkine/CXC3CL1 were revealed in the plasma of pharmacoresistant patients than in controls (p < 0.0704), and we determined an upward trend in CSF IL-8 levels (p < 0.08). No significant differences in CSF and plasma levels were detected between pharmacodependent patients and controls. CONCLUSION: Elevated CCL2/MCP-1 in CSF and plasma, elevated levels of fractalkine/CXC3CL1 in CSF, and a trend toward elevated IL-8 in the CSF of patients with pharmacoresistant epilepsy indicate these cytokines as potential biomarkers of epileptogenesis and pharmacoresistance. CCL2/MCP-1was detected in blood plasma; this assessment may be easily achieved in clinical practice without the invasiveness of a spinal tap. However, due to the complexity of neuroinflammation in epilepsy, further studies are warranted to confirm our findings.
Východiska: Prognóza pacientů s karcinomem kolorekta (colorectal cancer – CRC) závisí především na rozsahu onemocnění v době diagnózy, proto je brzký záchyt jedním z hlavních předpokladů úspěšné léčby. Současný výzkum ukazuje, že exozomální dlouhé nekódující RNA (lncRNA) jsou spojeny s rozvojem nádorových onemocnění. Jelikož jsou lncRNA často tkáňově specifické, jejich kvantifikace v exozomech se nabízí jako neinvazivní metoda pro včasnou detekci CRC. V naší práci jsme se zaměřili na optimalizaci protokolu pro analýzu exozomálních lncRNA z krevního séra pacientů s CRC jako potenciálních diagnostických biomarkerů. Materiál a metody: Exozomy byly izolovány pomocí gelové chromatografie ze 150 μl séra pacientů s CRC a zdravých dárců. Jejich kvalita a kvantita byla potvrzena elektronovou mikroskopií a analýzou dynamického rozptylu světla (dynamic light scattering – DLS) a proteinové markery byly detekovány metodou Western blot. Po izolaci RNA byly ze vzorků připraveny cDNA knihovny, které byly sekvenovány pomocí NextSeq 550. Výsledky: Úspěšně jsme izolovali exozomy a ověřili jsme jejich vlastnosti několika různými metodami. Knihovny byly připraveny ze všech vzorků i přes velmi nízký objem výchozího materiálu. Sekvenační data potvrzují přítomnost protein kódující (50 %) i nekódující RNA, kterou tvoří především lncRNA (28,2 %), pseudogeny (15,2 %) a další typy RNA (6,5 %). Výsledky dále ukázaly významně změněné hladiny některých lncRNA, na základě jejichž exprese bylo možné odlišit vzorky od pacientů s CRC od vzorků zdravých kontrol. Pomocí analýzy obohacení genové sady (gene set enrichment analysis – GSEA) jsme pozorovali významně obohacené třídy genů, které souvisejí s opravami DNA nebo regulací buněčného cyklu. Závěr: Naše pilotní data naznačují, že lncRNA představují významnou část RNA přítomné v exozomech a jejich rozdílné hladiny mají schopnost odlišit CRC pacienty od zdravých kontrol. Analýza obohacených genů zároveň prokázala významné zastoupení lncRNA podílejících se na regulaci buněčného cyklu a oprav DNA, což naznačuje jejich možné zapojení do procesů kancerogeneze. Výsledky je však třeba ověřit na větším souboru pacientů.
Background: The prognosis of patients with colorectal cancer (CRC) depends mainly on the extent of the disease at the time of diagnosis; therefore, early detection is one of the main prerequisites for successful treatment. Current research shows that exosomal long non-coding RNAs (lncRNAs) are associated with cancer development. As lncRNAs are often tissue specific, their quantification in exosomes is proposed as a non-invasive method for early detection of CRC. In this study, we aimed to optimize a protocol for analyzing exosomal lncRNAs from blood serum of CRC patients as potential diagnostic biomarkers. Material and methods: Exosomes were isolated by gel chromatography from 150 μl of serum of CRC patients and healthy donors. Their quality and quantity were confirmed by electron microscopy and dynamic light scattering (DLS) analysis; protein markers were detected by Western blot. After RNA isolation, cDNA libraries were prepared and sequenced using NextSeq 550. Results: We successfully isolated exosomes and verified them by several methods. Libraries were prepared from all samples despite very low volume of starting material. The sequencing data confirmed the presence of both protein-coding (50%) and non-coding RNAs, which consisted mainly of lncRNAs (28.2%), pseudogenes (15.2%) and other RNA types (6.5%). The results also showed significantly altered levels of some lncRNAs that could distinguish samples from CRC patients and healthy controls. Using gene set enrichment analysis (GSEA), we observed significantly enriched classes of genes related to DNA repair or cell cycle regulation. Conclusion: Our preliminary data suggest that lncRNAs represent a significant fraction of the RNA present in exosomes and that their distinct levels can separate CRC patients from healthy controls. The analysis of enriched genes also showed a significant representation of lncRNAs involved in cell cycle regulation and DNA repair, suggesting their possible involvement in cancerogenesis. However, the results need to be verified in a larger cohort of patients.
Recirculation of chronic lymphocytic leukemia (CLL) cells between the peripheral blood and lymphoid niches plays a critical role in disease pathophysiology, and inhibiting this process is one of the major mechanisms of action for B-cell receptor (BCR) inhibitors such as ibrutinib and idelalisib. Migration is a complex process guided by chemokine receptors and integrins. However, it remains largely unknown how CLL cells integrate multiple migratory signals while balancing survival in the peripheral blood and the decision to return to immune niches. Our study provided evidence that CXCR4/CD5 intraclonal subpopulations can be used to study the regulation of migration of CLL cells. We performed RNA profiling of CXCR4dimCD5bright vs CXCR4brightCD5dim CLL cells and identified differential expression of dozens of molecules with a putative function in cell migration. GRB2-associated binding protein 1 (GAB1) positively regulated CLL cell homing capacity of CXCR4brightCD5dim cells. Gradual GAB1 accumulation in CLL cells outside immune niches was mediated by FoxO1-induced transcriptional GAB1 activation. Upregulation of GAB1 also played an important role in maintaining basal phosphatidylinositol 3-kinase (PI3K) activity and the "tonic" AKT phosphorylation required to sustain the survival of resting CLL B cells. This finding is important during ibrutinib therapy, because CLL cells induce the FoxO1-GAB1-pAKT axis, which represents an adaptation mechanism to the inability to home to immune niches. We have demonstrated that GAB1 can be targeted therapeutically by novel GAB1 inhibitors, alone or in combination with BTK inhibition. GAB1 inhibitors induce CLL cell apoptosis, impair cell migration, inhibit tonic or BCR-induced AKT phosphorylation, and block compensatory AKT activity during ibrutinib therapy.
- MeSH
- adaptorové proteiny signální transdukční biosyntéza MeSH
- adenin analogy a deriváty farmakologie MeSH
- chronická lymfatická leukemie farmakoterapie metabolismus patologie MeSH
- forkhead box protein O1 metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- piperidiny farmakologie MeSH
- pohyb buněk * MeSH
- protoonkogenní proteiny c-akt metabolismus MeSH
- regulace genové exprese u leukemie * MeSH
- signální transdukce * MeSH
- upregulace * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
There is an emerging body of evidence that patients with chronic myeloid leukaemia (CML) may carry not only breakpoint cluster region-Abelson murine leukaemia viral oncogene homologue 1 (BCR-ABL1) kinase domain mutations (BCR-ABL1 KD mutations), but also mutations in other genes. Their occurrence is highest during progression or at failure, but their impact at diagnosis is unclear. In the present study, we prospectively screened for mutations in 18 myeloid neoplasm-associated genes and BCR-ABL1 KD in the following populations: bulk leucocytes, CD34+ CD38+ progenitors and CD34+ CD38- stem cells, at diagnosis and early follow-up. In our cohort of chronic phase CML patients, nine of 49 patients harboured somatic mutations in the following genes: six ASXL1 mutations, one SETBP1, one TP53, one JAK2, but no BCR-ABL1 KD mutations. In seven of the nine patients, mutations were detected in multiple hierarchical populations including bulk leucocytes at diagnosis. The mutation dynamics reflected the BCR-ABL1 transcript decline induced by treatment in eight of the nine cases, suggesting that mutations were acquired in the Philadelphia chromosome (Ph)-positive clone. In one patient, the JAK2 V617F mutation correlated with a concomitant Ph-negative myeloproliferative neoplasm and persisted despite a 5-log reduction of the BCR-ABL1 transcript. Only two of the nine patients with mutations failed first-line therapy. No correlation was found between the mutation status and survival or response outcomes.
