A near-infrared light-activatable Ru(ii)-coumarin photosensitizer active under hypoxic conditions.

Photodynamic therapy (PDT) represents a promising approach for cancer treatment. However, the oxygen dependency of PDT to generate reactive oxygen species (ROS) hampers its therapeutic efficacy, especially against hypoxic solid tumors. In addition, some photosensitizers (PSs) have dark toxicity and are only activatable with short wavelengths such as blue or UV-light, which suffer from poor tissue penetration. Herein, we developed a novel hypoxia-active PS with operability in the near-infrared (NIR) region based on the conjugation of a cyclometalated Ru(ii) polypyridyl complex of the type [Ru(C^N)(N^N)2] to a NIR-emitting COUPY dye. The novel Ru(ii)-coumarin conjugate exhibits water-solubility, dark stability in biological media and high photostability along with advantageous luminescent properties that facilitate both bioimaging and phototherapy. Spectroscopic and photobiological studies revealed that this conjugate efficiently generates singlet oxygen and superoxide radical anions, thereby achieving high photoactivity toward cancer cells upon highly-penetrating 740 nm light irradiation even under hypoxic environments (2% O2). The induction of ROS-mediated cancer cell death upon low-energy wavelength irradiation along with the low dark toxicity exerted by this Ru(ii)-coumarin conjugate could circumvent tissue penetration issues while alleviating the hypoxia limitation of PDT. As such, this strategy could pave the way to the development of novel NIR- and hypoxia-active Ru(ii)-based theragnostic PSs fuelled by the conjugation of tunable, low molecular-weight COUPY fluorophores.

Exploring Structure-Activity Relationships in Photodynamic Therapy Anticancer Agents Based on Ir(III)-COUPY Conjugates.

Photodynamic therapy holds great promise as a non-invasive anticancer tool against drug-resistant cancers. However, highly effective, non-toxic, and reliable photosensitizers with operability under hypoxic conditions remain to be developed. Herein, we took the advantageous properties of COUPY fluorophores and cyclometalated Ir(III) complexes to develop novel PDT agents based on Ir(III)-COUPY conjugates with the aim of exploring structure-activity relationships. The structural modifications carried out within the coumarin scaffold had a strong impact on the photophysical properties and cellular uptake of the conjugates. All Ir(III)-COUPY conjugates exhibited high phototoxicity under green light irradiation, which was attributed to the photogeneration of ROS, while remaining non-toxic in the dark. Among them, two hit conjugates showed excellent phototherapeutic indexes in cisplatin-resistant A2780cis cancer cells, both in normoxia and in hypoxia, suggesting that photoactive therapy approaches based on the conjugation of far-red/NIR-emitting COUPY dyes and transition metal complexes could effectively tackle in vitro acquired resistance to cisplatin.

Extranodal natural killer/T-cell lymphoma nasal type in a western population: Molecular profiling identifies new therapeutic targets.

(A) Correlation matrix of unsupervised co-regulated genes, based on the 208 genes included in the NanoString platform. Some of the clusters of co-regulated genes corresponded to the following: Inflammatory cells; Epstein-Barr virus; B-cells; Cytotoxic T-cells; T-cells; and Proliferation. (B) Analysis of genomic alterations by targeted sequencing. Distribution of mutations in the 62 analyzed genes. Rows correspond to sequenced genes, columns represent individual patients. Color coding: green, missense; blue, synonymous; pink, frameshift; violet, Indel; red, stop gained; yellow, UTR.

Improving Photodynamic Therapy Anticancer Activity of a Mitochondria-Targeted Coumarin Photosensitizer Using a Polyurethane-Polyurea Hybrid Nanocarrier.

Integration of photosensitizers (PSs) within nanoscale delivery systems offers great potential for overcoming some of the "Achiles' heels" of photodynamic therapy (PDT). Herein, we have encapsulated a mitochondria-targeted coumarin PS into amphoteric polyurethane-polyurea hybrid nanocapsules (NCs) with the aim of developing novel nanoPDT agents. The synthesis of coumarin-loaded NCs involved the nanoemulsification of a suitable prepolymer in the presence of a PS without needing external surfactants, and the resulting small nanoparticles showed improved photostability compared with the free compound. Nanoencapsulation reduced dark cytotoxicity of the coumarin PS and significantly improved in vitro photoactivity with red light toward cancer cells, which resulted in higher phototherapeutic indexes compared to free PS. Importantly, this nanoformulation impaired tumoral growth of clinically relevant three-dimensional multicellular tumor spheroids. Mitochondrial photodamage along with reactive oxygen species (ROS) photogeneration was found to trigger autophagy and apoptotic cell death of cancer cells.

COUPY Coumarins as Novel Mitochondria-Targeted Photodynamic Therapy Anticancer Agents.

Photodynamic therapy (PDT) for cancer treatment has drawn increased attention over the last decades. Herein, we introduce a novel family of low-molecular-weight coumarins as potential PDT anticancer tools. Through a systematic study with a library of 15 compounds, we have established a detailed structure-activity relationship rationale, which allowed the selection of three lead compounds exhibiting effective in vitro anticancer activities upon visible-light irradiation in both normoxia and hypoxia (phototherapeutic indexes up to 71) and minimal toxicity toward normal cells. Acting as excellent theranostic agents targeting mitochondria, the mechanism of action of the photosensitizers has been investigated in detail in HeLa cells. The generation of cytotoxic reactive oxygen species, which has been found to be a major contributor of the coumarins' phototoxicity, and the induction of apoptosis and/or autophagy have been identified as the cell death modes triggered after irradiation with low doses of visible light.

Unexpected photoactivation pathways in a folate-receptor-targeted trans-diazido Pt(IV) anticancer pro-drug.

A conjugate between a photoactive trans-diazido Pt(iv) pro-drug, trans,trans,trans-[Pt(N3)2(OH)2(py)2], and folic acid has been synthesized and fully characterized by high resolution ESI-MS, NMR and UV-vis spectroscopy. Photoactivation of the Pt-folate conjugate with visible light confirmed the generation of cytotoxic Pt(ii) species capable of binding to guanine nucleobases. Importantly, photoreduction of the Pt(iv) complex triggered the photodecomposition of the folate vector into a p-aminobenzoate-containing fragment and several pterin derivatives, including 6-formylpterin. Besides exhibiting high dark stability in physiological-like conditions, the Pt-folate conjugate was ca. 2× more photocytotoxic towards MCF-7 breast cancer cell line than its parent Pt(iv) complex with a high photoselectivity index (PI > 6.9). The higher photocytotoxicity of the conjugate may be a consequence of its higher cellular accumulation and of the generation of a set of different cytotoxic species, including Pt(ii) photoproducts and several pterin derivatives, which are known to generate ROS.

Bone marrow fibrosis, sequence variant of asxl1, and Sjögren syndrome: A case report.

Only proven pathogenic mutations associated with myeloid neoplasms are key to establish the clonal nature of the bone marrow fibrosis. In cases with genetic variants of uncertain meaning, the clinical picture may be required to rule out secondary causes.

Modulating Photostability and Mitochondria Selectivity in Far-Red/NIR Emitting Coumarin Fluorophores through Replacement of Pyridinium by Pyrimidinium.

Mitochondrial dysfunction has been associated with several human pathological conditions, including cancer, aging, and neurodegenerative diseases. Thus, the availability of selective fluorescent probes for mitochondria could play an important role in the future for monitoring cellular functions and disease progression. In this work, we have studied how the photophysical properties and subcellular accumulation of nonconventional coumarin-based COUPY fluorophores can be fine-tuned through replacement of the para-pyridinium moiety with several heterocycles. Among them, ortho,para-pyrimidinium substitution provided novel fluorophores with suitable photophysical properties for bioimaging applications, including emission in the far-red to NIR region, large Stokes' shifts, and high photostability. Furthermore, the compounds exhibited excellent cell membrane permeability in living cells and a higher selectivity for mitochondria compared with the parent COUPY fluorophores. Overall, these results provided useful insights into the development of novel mitochondria-targeted fluorescent probes based on small organic molecules, since higher selectivity for this organelle can be achieved through the replacement of conventional N-alkylated pyridinium moieties by the corresponding N-alkylated-ortho,para-pyrimidinium counterparts.

Ultrastructural, cytogenetic, and molecular findings in mast cell leukemia: Case report.

We report a de novo aleukemic form of MCL with a complex monosomic karyotype with LOH for multiple chromosomes and TP53 mutation. Additionally, whereas D816V KIT was not found, the c-Kit transmembrane domain p.M541L variant was detected which is the most common SNP of KIT gene in humans with controversial pathogenic role. In these cases, it is crucial to perform a rapid broad molecular study for an accurate diagnosis which could help to initiate targeted therapy.

Towards Novel Photodynamic Anticancer Agents Generating Superoxide Anion Radicals: A Cyclometalated IrIII Complex Conjugated to a Far-Red Emitting Coumarin.

Although cyclometalated IrIII complexes have emerged as promising photosensitizers for photodynamic therapy, some key drawbacks still hamper clinical translation, such as operability in the phototherapeutic window and reactive oxygen species (ROS) production efficiency and selectivity. In this work, a cyclometalated IrIII complex conjugated to a far-red-emitting coumarin, IrIII -COUPY, is reported with highly favourable properties for cancer phototherapy. IrIII -COUPY was efficiently taken up by HeLa cells and showed no dark cytotoxicity and impressive photocytotoxicity indexes after irradiation with green and blue light, even under hypoxia. Importantly, a clear correlation between cell death and intracellular generation of superoxide anion radicals after visible light irradiation was demonstrated. This strategy opens the door to novel fluorescent photodynamic therapy agents with promising applications in theragnosis.

Solid-Phase Approaches for Labeling Targeting Peptides with Far-Red Emitting Coumarin Fluorophores.

Fluorophores based on organic molecules hold great potential for ligand-targeted imaging applications, particularly those operating in the optical window in biological tissues. In this work, we have developed three straightforward solid-phase approaches based on amide-bond formation or a Cu(I)-catalyzed azide-alkyne click (CuAAC) reaction for labeling an octreotide peptide with far-red emitting coumarin-based COUPY dyes. First, the conjugatable versions of COUPY fluorophores incorporating the required functional groups (e.g., carboxylic acid, azide, or alkyne) were synthesized and characterized. All of them were found fully compatible with Fmoc/ tBu solid-phase peptide synthesis, which allowed for the labeling of octreotide either through amide-bond formation or by CuAAC reaction. A near quantitative conversion was obtained after only 1 h of reaction at RT when using CuSO4 and sodium ascorbate independently of the click chemistry approach used (azido-COUPY/alkynyl-peptide resin or alkynyl-COUPY/azido-peptide resin). COUPY-octreotide conjugates were found stable in cell culture medium as well as noncytotoxic in HeLa cells, and their spectroscopic and photophysical properties were found similar to those of their parent coumarin dyes. Finally, the potential bioimaging applications of COUPY-octreotide conjugates were demonstrated by confocal microscopy through the visualization of living HeLa cells overexpressing the somatostatin subtype-2 receptor.

Differential prognostic impact of GELTAMO-IPI in cell of origin subtypes of Diffuse Large B Cell Lymphoma as defined by the Hans algorithm.

The Grupo Español de Linfomas y Trasplantes de Médula Ósea International Prognostic Index (GELTAMO-IPI) stratifies four risk groups in diffuse large B cell lymphoma (DLBCL) patients treated with immunochaemotherapy: low (LR), low-intermediate (LIR), high-intermediate (HIR), and high (HR). The present study explores the effect of GELTAMO-IPI in the DLBCL subtypes defined by the immunohistochaemistry-based Hans algorithm, Germinal Centre B (GCB) and non-GCB. A multivariate Cox regression model including GELTAMO-IPI risk groups, cell of origin (COO) subtypes and their product was developed to evaluate interaction between the two variables. The COO subtype was available in 839 patients (380 GCB; 459 non-GCB) and both the GELTAMO-IPI and the COO subtype in 780 (353 GCB; 427 non-GCB). There were no differences in 5-year overall survival (OS) between the two subtypes. The Cox model revealed interaction between the GELTAMO-IPI risk groups and the COO subtypes (P = 0·005), indicating that GELTAMO-IPI has a different effect in the two subtypes. Three risk groups were stratified in both COO subtypes: in the GCB subtype, LR, LIR and the combined HIR+HR had 5-year OS of 100%, 75% and 52%, respectively. In the non-GCB subtype, LR, the combined LIR+HIR and HR had a 5-year OS of, 97%, 82% and 35% respectively. GELTAMO-IPI identifies a genuine poor outcome group of patients in the DLBCL non-GCB subtype.

Redesigning the Coumarin Scaffold into Small Bright Fluorophores with Far-Red to Near-Infrared Emission and Large Stokes Shifts Useful for Cell Imaging.

Among the palette of previously described fluorescent organic molecules, coumarins are ideal candidates for developing cellular and molecular imaging tools due to their high cell permeability and minimal perturbation of living systems. However, blue-to-cyan fluorescence emission is usually difficult in in vivo applications due to the inherent toxicity and poor tissue penetration of short visible light wavelengths. Here, we introduce a new family of coumarin-based fluorophores, nicknamed COUPY, with promising photophysical properties, including emission in the far-red/near-infrared (NIR) region, large Stokes shifts, high photostability, and excellent brightness. COUPY fluorophores were efficiently synthesized in only three linear synthetic steps from commercially available precursors, with the N-alkylation of a pyridine moiety being the key step at the end of the synthetic route, as it allows for the tuning of the photophysical properties of the resulting dye. Owing to their low molecular weights, COUPY dyes show excellent cell permeability and accumulate selectively in nucleoli and/or mitochondria of HeLa cells, as their far-red/NIR fluorescence emission is easily detected at a concentration as low as 0.5 µM after an incubation of only 20 min. We anticipate that these coumarin scaffolds will open a way to the development of novel coumarin-based far-red to NIR emitting fluorophores with potential applications for organelle imaging and biomolecule labeling.

A Comparison of RNA-Seq Results from Paired Formalin-Fixed Paraffin-Embedded and Fresh-Frozen Glioblastoma Tissue Samples.

The molecular classification of glioblastoma (GBM) based on gene expression might better explain outcome and response to treatment than clinical factors. Whole transcriptome sequencing using next-generation sequencing platforms is rapidly becoming accepted as a tool for measuring gene expression for both research and clinical use. Fresh frozen (FF) tissue specimens of GBM are difficult to obtain since tumor tissue obtained at surgery is often scarce and necrotic and diagnosis is prioritized over freezing. After diagnosis, leftover tissue is usually stored as formalin-fixed paraffin-embedded (FFPE) tissue. However, RNA from FFPE tissues is usually degraded, which could hamper gene expression analysis. We compared RNA-Seq data obtained from matched pairs of FF and FFPE GBM specimens. Only three FFPE out of eleven FFPE-FF matched samples yielded informative results. Several quality-control measurements showed that RNA from FFPE samples was highly degraded but maintained transcriptomic similarities to RNA from FF samples. Certain issues regarding mutation analysis and subtype prediction were detected. Nevertheless, our results suggest that RNA-Seq of FFPE GBM specimens provides reliable gene expression data that can be used in molecular studies of GBM if the RNA is sufficiently preserved.