Human Cancer Cell Cultures Research Articles

Anticancer therapeutic potential of genus Diospyros: From phytochemistry to clinical applications-A review.

The genus Diospyros has gained significant attention in the scientific community owing to its diverse bioactivities ascribed to specific bioactive constituents present in different species of this plant. Phytochemicals like flavonoids, terpenoids, and xanthones have been reported to be present in other Diospyros species responsible for their pharmacological properties. These compounds are well known for their diverse potent therapeutic potentials, such as antimicrobial, antioxidant, anti-inflammatory, and anticancer properties. This review enlightens the details of the Genus Diospyros, ranging from an overview of its species to an in-depth analysis of phytochemistry, ethnopharmacology, and their potential as anticancer agents. Different species, including Diospyros lotus, Diospyros kaki, Diospyros maritima, Diospyros mespiliformis, and Diospyros tricolor, presented with an enormous range of anticancer activities against human cancer cell cultures. Moreover, this review highlights the results of various invitro (antiproliferative, cytotoxic effects against), invivo (inhibition of tumor, apoptosis), and in silico (GLU234, GLU278, and LYS158 protein residues) studies, elucidating its preclinical anticancer potential. The anticancer potential displays inhibition of cellular proliferation, induction of apoptosis, and mitigation of angiogenesis. Furthermore, this review may elaborate the use of traditional knowledge, modern research, and potential therapeutic applications in the field of anticancer ethnopharmacology. As the modern-day research approaches novel alternatives to combat diseases like cancer, the Genus Diospyros may emerge as a promising avenue with the potential to yield innovative and effective therapeutic agents.

Carbon nanotubes conjugated with cisplatin activate different apoptosis signaling pathways in 2D and 3D-spheroid triple-negative breast cancer cell cultures: a comparative study

The type of experimental model for the in vitro testing of drug formulations efficiency represents an important tool in cancer biology, with great attention being granted to three-dimensional (3D) cultures as these offer a closer approximation of the clinical sensitivity of drugs. In this study, the effects induced by carboxyl-functionalized single-walled carbon nanotubes complexed with cisplatin (SWCNT–COOH–CDDP) and free components (SWCNT–COOH and CDDP) were compared between conventional 2D- and 3D-spheroid cultures of human breast cancer cells. The 2D and 3D breast cancer cultures were exposed to various doses of SWCNT–COOH (0.25–2 μg/mL), CDDP (0.158–1.26 μg/mL) and the same doses of SWNCT–COOH–CDDP complex for 24 and 48 h. The anti-tumor activity, including modulation of cell viability, oxidative stress, proliferation, apoptosis, and invasion potential, was explored by spectrophotometric and fluorometric methods, immunoblotting, optical and fluorescence microscopy. The SWCNT–COOH–CDDP complex proved to have high anti-cancer efficiency on 2D and 3D cultures by inhibiting cell proliferation and activating cell death. A dose of 0.632 μg/mL complex triggered different pathways of apoptosis in 2D and 3D cultures, by intrinsic, extrinsic, and reticulum endoplasmic pathways. Overall, the 2D cultures showed higher susceptibility to the action of complex compared to 3D cultures and SWCNT–COOH–CDDP proved enhanced anti-tumoral activity compared to free CDDP.

A novel flexible electrochemical sensor with multi-level carbon nano-array for in-situ real-time monitoring of H2O2 secreted by cells

Developing ultra-sensitive sensors for in-situ real-time monitoring of low-level cell secretions is of paramount importance in understanding various biological processes, and it is still a challenge. Herein, for the first time, we constructed a novel flexible Au-Co@C-CNT/CC sensing electrode with nano-array structures by in-situ synthesis, catalytic pyrolysis and electrochemical deposition, and successfully realized the real-time monitoring of H2O2 released by cells. The 3D leaf-like structure of the electrode not only provided a large electroactive surface area (7.66 cm2) but also exhibited a microvilli structure of carbon nanotubes (CNT) on the surface, which further enhanced the presence of active sites. Moreover, the Co-Nx sites generated by catalytic pyrolysis can promote the adsorption and catalysis of H2O2 on the surface of sensing electrode. The introduction of Au NPs facilitated the transfer of electrons, thereby enhancing the electrical conductivity and electrocatalytic performance of Au-Co@C-CNT/CC. Benefiting from these unique superiorities, this sensing platform displayed excellent performance for H2O2 determination with a wide linear range (10–27080 μM) and low detection limit (0.13 μM). Notably, the good biocompatibility and flexibility of this sensing platform allowed direct culture of human lung cancer cells (A549 cells) on the electrodes, which can realize dynamic in-situ real-time detection of H2O2 released by cells, showing great potential in the diagnosis of related diseases.

A novel dihydroacridine derivative targets epidermal growth factor receptor-expressing cancer cells in vitro and in vivo.

Small molecules are considered a source of novel medicines targeting carcinogenic intracellular pathways including epidermal growth factor receptor (EGFR) signaling. The main goal of the study is to assess whether LHT-17-19 could be considered an effective target molecule against EGFR-expressing tumor cells in silico, in vitro, and in vivo. This was an in vivo, ex vivo, and in vivo experimental study. LHT-17-19 affinity to EGFR's kinase domain was assessed by the ligand's molecular docking. EGFR-expressing Hs746T human gastric cancer cell culture and patient-derived organoid (PDO) model of EGFR-positive breast cancer (BC) were used for in vitro assessment of the molecule anticancer property. IC50 and GI50 indexes were estimated using MTT- and MTS-based tests, respectively. Anticancer activity of LHT-17-19 against EGFR-expressing mutant lung carcinoma was studied on patient-derived xenograft (PDX) model established in 10 humanized BALB/c male mice. Continuous variables were presented as a mean ± standard deviation. Intergroup differences were assessed by two-way t-test. Kaplan-Meier's curves were used for survival analysis. High affinity of LHT-17-19 for the EGFR kinase domain with dG score -7.9 kcal/mol, EDoc-5.45 kcal/mol, and Ki 101.24 uM was due to intermolecular π-σ bonds formation and the ligand intramolecular transformation. LHT-17-19 induced anti-EGFR-expressing gastric cancer cells cytotoxicity with IC50 0.32 µM (95% confidence interval [CI] 0.11-0.54 µM). The derivative inhibited growth of EGFR-expressing BC PDO with GI50 16.25 µM (95% CI 4.44-28.04 µM). 2 mg/kg LHT-17-19 intravenously daily during 7 days inhibited PDX tumor growth and metastatic activity, prolonged animals' survival, and eliminated EGFR-mutant lung cancer cells from residual tumor's node. LHT-17-19 may be considered a molecular platform for further search of promising molecules, EGFR-expressing cancer cell inhibitors.

Ligand-gated ion channels as potential biomarkers for ADT-mediated cognitive decline in prostate cancer patients.

Men with prostate cancer are at increased risk of developing cognitive decline by the use of second-generation androgen signaling inhibitors. To date, reliable and sensitive biomarkers that could distinguish men at high risk of cognitive dysfunction under androgen deprivation therapy (ADT) have not been characterized. We used high-throughput transcriptional profiling utilizing human prostate cancer cell culture models mimicking ADT, biomarker selection using minimal common oncology data elements-cytoscape, and bioinformatic analyses employing Advaita® iPathwayGuide and DisGeNET for identification of disease-related gene associations. Validation analysis of genes was performed on brain neuronal and glial cells by quantitative real-time polymerase chain reaction assay. Our systematic analysis of androgen deprivation-associated genes involved multiple biological processes, including neuroactive ligand-receptor interaction, axon guidance, cytokine-cytokine receptor interaction, and metabolic and cancer signaling pathways. Genes associated with neuroreceptor ligand interaction, including gamma-aminobutyric acid (GABA) A and B receptors and nuclear core proteins, were identified as top upstream regulators. Functional enrichment and protein-protein interaction network analysis highlighted the role of ligand-gated ion channels (LGICs) and their receptors in cognitive dysfunction. Gene-disease association assigned forgetfulness, intellectual disability, visuospatial deficit, bipolar disorder, and other neurocognitive impairment with upregulation of type-1 angiotensin II receptor, brain-derived neurotrophic factor, GABA type B receptor subunit 2 (GABBR2), GABRA3, GABRA5, GABRB1, glycine receptor beta, glutamate ionotropic receptor N-methyl-D-aspartate receptor (NMDA) type subunit 1, glutamate ionotropic receptor NMDA type subunit 2D, 5-hydroxytryptamine receptor 1D, interferon beta 1, and nuclear receptor subfamily 3 group C member 1 as top differentially expressed genes. Validation studies of brain glial cells, neurons, and patients on ADT demonstrated the association of these genes with cognitive decline. Our findings highlight LGICs as potential biomarkers for ADT-mediated cognitive decline. Further validation of these biomarkers may lead to future practical clinical use.

Drug-delivery and biological activity in colorectal cancer of a supramolecular porous material assembled from heptameric chromium-copper-adenine entities.

The therapeutic application of drugs often faces challenges due to non-specific distribution, inadequate dosification and degradation, which limits their efficacy. Two primary strategies are employed to overcome these issues: the use of derivatives of the active substances and incorporation of those into porous materials. The latter, involving materials such as zeolites, metal-organic frameworks (MOFs), and hydrogels, has shown promising results in protecting the active ingredients from degradation and enabling a controlled release. This study focuses on supramolecular metal-organic frameworks (SMOFs), which leverage supramolecular interactions for enhanced pore size control. [Cu6Cr(μ-adeninato-κN3:κN9)6(μ3-OH)6(μ-OH2)6](SO4)1.5·nH2O (Cu6Cr) was chosen for its flexible porous structure, water-stability, and paramagnetic properties. Magnetic sustentation studies showed that this compound was able to capture several drug molecules: 5-fluorouracil (5-FU), 5-aminosalicylic acid (5-ASA), 4-aminosalicylic acid (4-ASA) and theophylline (THEO). Their release follows a pseudo-first-order kinetics with desorption half-lives ranging from 2.2 to 4.7 hours. In this sense, a novel approach is proposed using bulkier raffinose and cholesterol as pore-blocking molecules. Cholesterol exhibited the best performance as a blocking molecule increasing the desorption half-life up to 8.2 hours. Cytotoxicity and RNA-seq transcriptomic assays carried out on human colorectal cancer cell cultures showed, on one hand, that the Cu6Cr porous material exhibits a proliferative effect, probably coming from the over-expression of MIR1248 and SUMO2 genes, and on the other hand, that there is a delay in the emergence of the cytotoxicity of 5-FU as expected for a slower release.

Synthesis of Cytotoxic Quino[4,3-b]carbazole Frameworks through an Intramolecular Diels-Alder Reaction.

An intramolecular Diels-Alder reaction of positionally isomeric indole-2/3-phenylvinyl-N-alkynylated (N-phenylsulfonyl)amines has been successfully exploited for the synthesis of quino[4,3-b]carbazole and its analogues. This reaction proceeds through a [4 + 2] cycloaddition followed by elimination and deprotection of phenylsulfonyl units to afford the quinocarbazoles in moderate to good yields. The reaction features a broad substrate scope and remarkable functional group forbearance. A preliminary in vitro cytotoxicity evaluation of representative quino[4,3-b]carbazoles was performed against NCI-H460 human cancer cell culture. Among the quino[4,3-b]carbazoles evaluated, five of the fluorine-containing quinocarbazoles displayed nano molar range (0.8-2.0 nm) GI50 values. The UV-vis and fluorescence spectral studies of representative quinocarbazoles were also performed. Like ellipticine, four of the quinocarbazoles displayed dual emissions confirming the existence of p-quinonoid like tautomeric forms in a polar protic solvent.

Modulation of Expression of Drug Metabolizing Enzymes and Augmentation of Anti-cancer Drug Effects: Through Epigenetics and Three-dimensional Cancer Cell Culture Systems

Since commencing my role as a professor in a newly established Department of Pharmacodynamics and Molecular Genetics at the School of Pharmacy, Iwate Medical University, on April 1, 2007, my research has focused on modifying gene expression of cytochrome P-450 (CYP) in established human colon cancer cells. Additionally, I have been investigating methods to enhance the anti-tumor effects of irinotecan (CPT-11) and 5-fluorouracil (5-FU) using epigenetic modifying inhibitors of DNA methyltransferase and histone deacetylase. Treating colon cancer cells with a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (DAC), led to elevated expression levels of CYP1B1 and CYP3A4 through demethylation of the promoter regions of related genes. Furthermore, the administration of DAC and the histone deacetylase inhibitor depsipeptide [(DEP), an anti-cancer drug romidepsin] significantly increased the cellular sensitivities of human colon cancer cells to CPT-11 and 5-FU, respectively. Remarkably, DAC treatment also increased colon cancer cell sensitivity to SN-38, an active metabolite of CPT-11, through the suppression of the anti-apoptotic protein Bcl-2. DEP increased colon cancer cell sensitivity to 5-FU in association with increased expressions of tumor-suppressor p21 and major histocompatibility complex class II genes. Another facet of my research is centered around understanding the gene regulatory mechanisms of the CYP1 family through aryl hydrocarbon receptors (AhR)s under glucose-deprivation stress and in three-dimensional (3D) culture systems of human solid tumor cells. In the 3D culture of human liver cancer cells, I found Pregnane X Receptor being implicated in the regulation of CYP1A2, which aligns with the in vivo mode of CYP1A2 expression.

ICVD Polymer Thin Film Bio‐Interface‐Performance for Fibroblasts, Cancer‐Cells, and Viruses Connected to Their Functional Groups and In Silico Studies

AbstractThin polymer coatings are used to improve the interface between biological species and functional materials. Their interaction is significantly influenced by the functional groups and roughness of the polymer film and prediction of the interaction is thus of great interest. However, for conventional polymer films, this cannot be examined independently because of the interplay of defects, residual solvent molecules, roughness, and functional groups. Solvent‐free polymer films prepared by initiated chemical vapor deposition (iCVD) exhibit conformal, defect‐free characteristics and enable precise tailoring of the functional groups. This facilitates to isolate the contribution of functional groups on the bio‐interface performance. Consequently, in silico studies can enable a prediction of ligand interaction in anti‐viral activity for SARS‐CoV‐2 based on defined polymer and key protein structures. Furthermore, the cell viability of human fibroblasts can be traced back to the functional groups of the repeating units. For human liver cancer cell culture, it turns out that more sophisticated models are needed. The insilico‐iCVD approach can enable precise tailoring of complex polymer films optimized for the respective interfaces. In addition, this first big scan of the bio‐interface performance of iCVD films enables a solid starting point in areas like anticancer, antiviral, and biocompatibility for future studies.

Muscarinic receptor agonist-induced βPix binding to β-catenin promotes colon neoplasia

M3 muscarinic receptors (M3R) modulate β-catenin signaling and colon neoplasia. CDC42/RAC guanine nucleotide exchange factor, βPix, binds to β-catenin in colon cancer cells, augmenting β-catenin transcriptional activity. Using in silico, in vitro, and in vivo approaches, we explored whether these actions are regulated by M3R. At the invasive fronts of murine and human colon cancers, we detected co-localized nuclear expression of βPix and β-catenin in stem cells overexpressing M3R. Using immunohistochemistry, immunoprecipitation, proximity ligand, and fluorescent cell sorting assays in human tissues and established and primary human colon cancer cell cultures, we detected time-dependent M3R agonist-induced cytoplasmic and nuclear association of βPix with β-catenin. βPix knockdown attenuated M3R agonist-induced human colon cancer cell proliferation, migration, invasion, and expression of PTGS2, the gene encoding cyclooxygenase-2, a key player in colon neoplasia. Overexpressing βPix dose-dependently augmented β-catenin binding to the transcription factor TCF4. In a murine model of sporadic colon cancer, advanced neoplasia was attenuated in conditional knockout mice with intestinal epithelial cell deficiency of βPix. Expression levels of β-catenin target genes and proteins relevant to colon neoplasia, including c-Myc and Ptgs2, were reduced in colon tumors from βPix-deficient conditional knockout mice. Targeting the M3R/βPix/β-catenin axis may have therapeutic potential.

Biodistribution and toxicity assessment of methoxyphenyl phosphonium carbosilane dendrimers in 2D and 3D cell cultures of human cancer cells and zebrafish embryos

The consideration of human and environmental exposure to dendrimers, including cytotoxicity, acute toxicity, and cell and tissue accumulation, is essential due to their significant potential for various biomedical applications. This study aimed to evaluate the biodistribution and toxicity of a novel methoxyphenyl phosphonium carbosilane dendrimer, a potential mitochondria-targeting vector for cancer therapeutics, in 2D and 3D cancer cell cultures and zebrafish embryos. We assessed its cytotoxicity (via MTT, ATP, and Spheroid growth inhibition assays) and cellular biodistribution. The dendrimer cytotoxicity was higher in cancer cells, likely due to its specific targeting to the mitochondrial compartment. In vivo studies using zebrafish demonstrated dendrimer distribution within the vascular and gastrointestinal systems, indicating a biodistribution profile that may be beneficial for systemic therapeutic delivery strategies. The methoxyphenyl phosphonium carbosilane dendrimer shows promise for applications in cancer cell delivery, but additional studies are required to confirm these findings using alternative labelling methods and more physiologically relevant models. Our results contribute to the growing body of evidence supporting the potential of carbosilane dendrimers as vectors for cancer therapeutics.

Synthesis of 1-(2-Hydroxyphenyl)- and (3,5-Dichloro-2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic Acid Derivatives as Promising Scaffolds for the Development of Novel Antimicrobial and Anticancer Agents.

Increasing antimicrobial resistance among Gram-positive pathogens and pathogenic fungi remains one of the major public healthcare threats. Therefore, novel antimicrobial candidates and scaffolds are critically needed to overcome resistance in Gram-positive pathogens and drug-resistant fungal pathogens. In this study, we explored 1-(2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic acid and its 3,5-dichloro-2-hydroxyphenyl analogue for their in vitro antimicrobial activity against multidrug-resistant pathogens. The compounds showed structure-dependent antimicrobial activity against Gram-positive pathogens (S. aureus, E. faecalis, C. difficile). Compounds 14 and 24b showed promising activity against vancomycin-intermediate S. aureus strains, and favorable cytotoxic profiles in HSAEC-1 cells, making them attractive scaffolds for further development. 5-Fluorobenzimidazole, having a 3,5-dichloro-2-hydroxyphenyl substituent, was found to be four-fold, and hydrazone, with a thien-2-yl fragment, was two-fold stronger than clindamycin against methicillin resistant S. aureus TCH 1516. Moreover, hydrazone, bearing a 5-nitrothien-2-yl moiety, showed promising activity against three tested multidrug-resistant C. auris isolates representing major genetic lineages (MIC 16 µg/mL) and azole-resistant A. fumigatus strains harboring TR34/L98H mutations in the CYP51A gene. The anticancer activity characterization demonstrated that the 5-fluorobenzimidazole derivative with a 3,5-dichloro-2-hydroxyphenyl substituent showed the highest anticancer activity in an A549 human pulmonary cancer cell culture model. Collectively these results demonstrate that 1-(2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic acid derivatives could be further explored for the development of novel candidates targeting Gram-positive pathogens and drug-resistant fungi.

Long-term organoid culture of a small intestinal neuroendocrine tumor.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are rare and highly heterogeneous neoplasms whose incidence has markedly increased over the last decades. A grading system based on the tumor cells' proliferation index predicts high-risk for G3 NETs. However, low-to-intermediate grade (G1/G2) NETs have an unpredictable clinical course that varies from indolent to highly malignant. Cultures of human cancer cells enable to perform functional perturbation analyses that are instrumental to enhance our understanding of cancer biology. To date, no tractable and reliable long-term culture of G1/G2 NET has been reported to permit disease modeling and pharmacological screens. Here, we report of the first long-term culture of a G2 metastatic small intestinal NET that preserves the main genetic drivers of the tumor and retains expression patterns of the endocrine cell lineage. Replicating the tissue, this long-term culture showed a low proliferation index, and yet it could be propagated continuously without dramatic changes in the karyotype. The model was readily available for pharmacological screens using targeted agents and as expected, showed low tumorigenic capacity in vivo. Overall, this is the first long-term culture of NETs to faithfully recapitulate many aspects of the original neuroendocrine tumor.

Anticancer Activity of Sunitinib Analogues in Human Pancreatic Cancer Cell Cultures under Normoxia and Hypoxia

Pancreatic cancer remains one of the deadliest cancer types. It is usually characterized by high resistance to chemotherapy. However, cancer-targeted drugs, such as sunitinib, recently have shown beneficial effects in pancreatic in vitro and in vivo models. Therefore, we chose to study a series of sunitinib derivatives developed by us, that were proven to be promising compounds for cancer treatment. The aim of our research was to evaluate the anticancer activity of sunitinib derivatives in human pancreatic cancer cell lines MIA PaCa-2 and PANC-1 under normoxia and hypoxia. The effect on cell viability was determined by the MTT assay. The compound effect on cell colony formation and growth was established by clonogenic assay and the activity on cell migration was estimated using a 'wound healing' assay. Six out of 17 tested compounds at 1 µM after 72 h of incubation reduced cell viability by 90% and were more active than sunitinib. Compounds for more detailed experiments were chosen based on their activity and selectivity towards cancer cells compared to fibroblasts. The most promising compound EMAC4001 was 24 and 35 times more active than sunitinib against MIA PaCa-2 cells, and 36 to 47 times more active against the PANC-1 cell line in normoxia and hypoxia. It also inhibited MIA PaCa-2 and PANC-1 cell colony formation. Four tested compounds inhibited MIA PaCa-2 and PANC-1 cell migration under hypoxia, but none was more active than sunitinib. In conclusion, sunitinib derivatives possess anticancer activity in human pancreatic adenocarcinoma MIA PaCa-2 and PANC-1 cell lines, and they are promising for further research.

Induction of damage to the dna structure of tumor cells by aurum polyacrylate

The ability of aurum polyacrylate (aurumacryl) to cause changes in the DNA structure of tumor cells (culture of MCF-7 human breast cancer cells) was studied. It was established that the molecular mechanism under- lying the effects of aurumacryl on tumor cells is associated with the induction of single-strand breaks and cross-links in the DNA molecule. At the same time, it was shown that aurumacryl can reduce the number of spontaneous, or irradiated, DNA double-strand breaks in MCF-7 cells.

Methylthioacetic acid, a derivative of aroma compounds from Cucumis melo var. conomon dose-dependently triggers differentiation and apoptosis of RCM-1 human colorectal cancer cells.

Methylthioacetic acid (MTA) is an acid-hydrolyzed derivative of a natural aroma compound, methylthioacetic acid ethyl ester isolated from Cucumis melo var. conomon (Katsura-uri, Japanese Picking Melon), and induces a villiform-like structure dome in RCM-1 human colorectal cancer cell culture. Thus far, the physiological and molecular properties of MTA-mediated dome formation remain unknown. Herein, MTA (not more than 2 mM) was demonstrated to differentiate the unorganized cell mass into the dome in RCM-1 cell culture by disclosing the correlation between dome formation and several intestinal differentiation markers such as alkaline phosphatase activity and the protein levels of dipeptidyl peptidase 4, villin, and Krüppel-like factor 4. Dome formation in RCM-1 cell culture was additively enhanced by the simultaneous administration of MTA and butyric acid (BA), suggesting that MTA directs the differentiation of RCM-1 cells, potentially through the same or similar pathway(s) shared with BA. Notably, a high dose of MTA (2 mM or more) elevated several apoptosis markers, such as DNA fragmentation, caspase-3/7 activity, and cleavage of poly(ADP-ribose) polymerase. Altogether, in addition to RCM-1 cell differentiation, MTA triggers apoptosis. These results indicate that MTA is a potential anticarcinogenic agent applicable in differentiation therapy and traditional chemotherapy against colorectal cancers.

Antenna-enhanced mid-infrared detection of extracellular vesicles derived from human cancer cell cultures

BackgroundExtracellular Vesicles (EVs) are sub-micrometer lipid-bound particles released by most cell types. They are considered a promising source of cancer biomarkers for liquid biopsy and personalized medicine due to their specific molecular cargo, which provides biochemical information on the state of parent cells. Despite this potential, EVs translation process in the diagnostic practice is still at its birth, and the development of novel medical devices for their detection and characterization is highly required.ResultsIn this study, we demonstrate mid-infrared plasmonic nanoantenna arrays designed to detect, in the liquid and dry phase, the specific vibrational absorption signal of EVs simultaneously with the unspecific refractive index sensing signal. For this purpose, EVs are immobilized on the gold nanoantenna surface by immunocapture, allowing us to select specific EV sub-populations and get rid of contaminants. A wet sample-handling technique relying on hydrophobicity contrast enables effortless reflectance measurements with a Fourier-transform infrared (FTIR) spectro-microscope in the wavelength range between 10 and 3 µm. In a proof-of-principle experiment carried out on EVs released from human colorectal adenocarcinoma (CRC) cells, the protein absorption bands (amide-I and amide-II between 5.9 and 6.4 µm) increase sharply within minutes when the EV solution is introduced in the fluidic chamber, indicating sensitivity to the EV proteins. A refractive index sensing curve is simultaneously provided by our sensor in the form of the redshift of a sharp spectral edge at wavelengths around 5 µm, where no vibrational absorption of organic molecules takes place: this permits to extract of the dynamics of EV capture by antibodies from the overall molecular layer deposition dynamics, which is typically measured by commercial surface plasmon resonance sensors. Additionally, the described metasurface is exploited to compare the spectral response of EVs derived from cancer cells with increasing invasiveness and metastatic potential, suggesting that the average secondary structure content in EVs can be correlated with cell malignancy.ConclusionsThanks to the high protein sensitivity and the possibility to work with small sample volumes—two key features for ultrasensitive detection of extracellular vesicles- our lab-on-chip can positively impact the development of novel laboratory medicine methods for the molecular characterization of EVs.Graphical

A Signaling Crosstalk Links SNAIL to the 37/67 kDa Laminin-1 Receptor Ribosomal Protein SA and Regulates the Acquisition of a Cancer Stem Cell Molecular Signature in U87 Glioblastoma Neurospheres.

Background: Three-dimensional in vitro neurospheres cultures recapitulate stemness features associated with poor clinical outcome in glioblastoma patients. They are commonly used to address brain cancer stem cell (CSC) signal transducing biology that regulates spheroids formation and stemness phenotype, and to assess the in vitro pharmacological impact of chemotherapeutic drugs. Objective: Here, we addressed the role of a new signaling axis involved in the regulation of in vitro spheroids formation and assessed the chemopreventive ability of diet-derived epigallocatechin gallate (EGCG) to impact the processes that govern the acquisition of spheroids CSC stemness traits. Methods: Neurospheres were generated from adherent human U87 glioblastoma cancer cell cultures under conditions that recapitulate stemness features. Total RNA and protein lysates were isolated for gene expression by RT-qPCR and protein expression by immunoblot. Transcriptomic analysis was performed through RNA-Seq. Results: Compared to their parental adherent cells, tumorspheres expressed increased levels of the CSC markers NANOG, SOX2, PROM1 (CD133), as well as of the epithelial-to-mesenchymal transition (EMT) markers Fibronectin, SNAI1, and 37/67 kDa laminin-1 receptor ribosomal protein SA (RPSA). Increased PROM1, SOX2, Fibronectin, and RPSA transcripts level were also observed in clinical grade IV glioblastoma tissues compared to normal tissue. EGCG treatment reduced dose-dependently tumorspheres size and inhibited the transcriptional regulation of those genes. An apoptotic signature was also found in spheroids with increased signal transducing events involving GSK3α/β, RSK, and CREB. These were repressed upon RPSA gene silencing and partially by SNAI1 silencing. Conclusion: This work highlights a signaling axis linking RPSA upstream of SNAIL in neurospheres genesis and supports the chemopreventive impact that diet-derived EGCG may exert on the acquisition of CSC traits.

On the mechanisms of photodynamic action of photosensitizers based on polycationic derivatives of synthetic bacteriochlorin against human lung cancer cells A549 (in vitro study)

BackgroundOne of the tasks of anticancer photodynamic therapy is increasing the efficacy of treatment of cancer nodes with large (clinically relevant) sizes using near-infrared photosensitizers (PS). We study the photodynamic action against A549 human lung cancer cells using PS based on polycationic derivatives of synthetic bacteriochlorin. MethodsThe efficacy and mechanisms of the photodynamic action of PS based on polycationic derivatives of synthetic bacteriochlorin against A549 lung cancer cells were studied in vitro using immunocytochemical and morphological methods. ResultsIt was found that PS based on tetracationic and octacationic derivatives of synthetic bacteriochlorin induce necrosis, apoptosis, decreasing of proliferative and mitotic activity, as well as reducing the number of ALDH1-positive cancer cells with signs of stem cells in A549 human lung cancer cell culture. The IC50 values (concentration of a PS that reduces cells survival by 50%) were about 0.69 μM for tetracationic PS and 0.57 μM for octacationic PS under irradiation at 30 J/cm2 while in the “dark” control they were higher than 100 μM for both PSs. ConclusionsPhotosensitizers based on polycationic derivatives of synthetic bacteriochlorin have high phototoxicity against A549 cancer cells caused by the induction of necrosis and apoptosis of cancer cells, including cells with signs of stemness, and a sharp decrease of mitotic and proliferative activity.

Combination of LIGHT (TNFSF14)-Armed Myxoma Virus Pre-Loaded into ADSCs and Gemcitabine in the Treatment of Experimental Orthotopic Murine Pancreatic Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly neoplasm. Oncolytic viruses have tumorolytic and immune response-boosting effects and present great potential for PDAC management. We used LIGHT-armed myxoma virus (vMyx-LIGHT) loaded ex vivo into human adipose-derived mesenchymal stem cells (ADSCs) to evaluate murine PDAC treatment in conjunction with gemcitabine (GEM). The cytotoxicity of this treatment was confirmed in vitro using human and murine pancreatic cancer cell cultures, which were more sensitive to the combined approach and largely destroyed. Unlike cancer cells, ADSCs sustain significant viability after infection. The in vivo administration of vMyx-LIGHT-loaded ADSCs and gemcitabine was evaluated using immunocompetent mice with induced orthotopic PDAC lesions. The expression of virus-encoded LIGHT increased the influx of T cells to the tumor site. Shielded virus followed by gemcitabine improved tumor regression and survival. The addition of gemcitabine slightly compromised the adaptive immune response boost obtained with the shielded virus alone, conferring no survival benefit. ADSCs pre-loaded with vMyx-LIGHT allowed the effective transport of the oncolytic construct to PDAC lesions and yielded significant immune response; additional GEM administration failed to improve survival. In view of our results, the delivery of targeted/shielded virus in combination with TGF-β ablation and/or checkpoint inhibitors is a promising option to improve the therapeutic effects of vMyx-LIGHT/ADSCs against PDAC in vivo.