Insight into the Course of the Ferrier Rearrangement Used to Obtain Untypical Diosgenyl Saponins.

The Ferrier rearrangement was utilized to obtain 2,3-unsaturated diosgenyl glycosides. This reaction proceeded with high stereoselectivity, yielding mostly saponins with an α configuration (hexoses) or predominantly with a ß configuration (pentoses). The diversity of the glycals used and the glycosides obtained enabled a deep discussion of the Ferrier rearrangement mechanism. The mechanism was supported by DFT calculations concerning the intermediate ions. It was concluded that the vinylogous anomeric effect may influence the reactivity of the glycals. Two possible Ferrier rearrangement intermediates, dioxolenium and allyloxycarbenium ions, were hypothesized to exist in thermodynamic equilibrium that shifted toward the former. The allyloxycarbenium ion participates in the final rearrangement step and determines the reaction regioselectivity. Furthermore, the conformational stability of the 2,3-unsaturated pyranose ring determines the stereoselectivity of the reaction. Factors influencing this stability, as well as the NMR data enabling recognition of the 0H5 and 5H0 conformations, were identified. Chemoselective hydrogenation of 2,3-unsaturated diosgenyl glycosides provided a series of 2,3-dideoxy analogues. The anticancer, hemolytic, and antibacterial activities of the synthesized saponins are presented alongside a discussion of the structure-activity relationships.

Development of potent and effective SARS-CoV-2 main protease inhibitors based on maleimide analogs for the potential treatment of COVID-19.

In the present work, we report a new series of potent SARS-CoV-2 Main Protease (Mpro) inhibitors based on maleimide derivatives. The inhibitory activities were tested in an enzymatic assay using recombinant Mpro (3CL Protease from coronavirus SARS-CoV-2). Within the set of new Mpro inhibitors, 6e demonstrated the highest activity in the enzymatic assay with an IC50 value of 8.52 ± 0.44 µM. The IC50 value for Nirmatrelvir (PF-07321332, used as a reference) was 0.84 ± 0.37 µM. The cytotoxic properties were determined in the MTT assay using MRC-5 and HEK-293 cell lines. In the course of the investigation, we found that the newly obtained maleimide derivatives are not substantially cytotoxic (IC50 values for most compounds were above 200 µM).

Role of extracellular vesicles in cancer-specific interactions between tumour cells and the vasculature.

Cancer progression impacts and exploits the vascular system in several highly consequential ways. Among different types of vascular cells, blood cells and mediators that are engaged in these processes, endothelial cells are at the centre of the underlying circuitry, as crucial constituents of angiogenesis, angiocrine stimulation, non-angiogenic vascular growth, interactions with the coagulation system and other responses. Tumour-vascular interactions involve soluble factors, extracellular matrix molecules, cell-cell contacts, as well as extracellular vesicles (EVs) carrying assemblies of molecular effectors. Oncogenic mutations and transforming changes in the cancer cell genome, epigenome and signalling circuitry exert important and often cancer-specific influences upon pathways of tumour-vascular interactions, including the biogenesis, content, and biological activity of EVs and responses of cancer cells to them. Notably, EVs may carry and transfer bioactive, oncogenic macromolecules (oncoproteins, RNA, DNA) between tumour and vascular cells and thereby elicit unique functional changes and forms of vascular growth and remodeling. Cancer EVs influence the state of the vasculature both locally and systemically, as exemplified by cancer-associated thrombosis. EV-mediated communication pathways represent attractive targets for therapies aiming at modulation of the tumour-vascular interface (beyond angiogenesis) and could also be exploited for diagnostic purposes in cancer.

Intramolecular Proton Transfer in the Radical Anion of Cytidine Monophosphate Sheds Light on the Sensitivities of Dry vs Wet DNA to Electron Attachment-Induced Damage.

Single-strand breaks (SSBs) induced via electron attachment were previously observed in dry DNA under ultrahigh vacuum (UHV), while hydrated electrons were found not able to induce this DNA damage in an aqueous solution. To explain these findings, crossed electron-molecular beam (CEMB) and anion photoelectron spectroscopy (aPES) experiments coupled to density functional theory (DFT) modeling were used to demonstrate the fundamental importance of proton transfer (PT) in radical anions formed via electron attachment. Three molecular systems were investigated: 5'-monophosphate of 2'-deoxycytidine (dCMPH), where PT in the electron adduct is feasible, and two ethylated derivatives, 5'-diethylphosphate and 3',5'-tetraethyldiphosphate of 2'-deoxycytidine, where PT is blocked due to substitution of labile protons with the ethyl residues. CEMB and aPES experiments confirmed the cleavage of the C3'/C5'-O bond as the main dissociation channel related to electron attachment in the ethylated derivatives. In the case of dCMPH, however, electron attachment (in the aPES experiments) yielded its parent (intact) radical anion, dCMPH-, suggesting that its dissociation was inhibited. The aPES-measured vertical detachment energy of the dCMPH- was found to be 3.27 eV, which agreed with its B3LYP/6-31++G(d,p)-calculated value and implied that electron-induced proton transfer (EIPT) had occurred during electron attachment to the dCMPH model nucleotide. In other words, EIPT, subduing dissociation, appeared to be somewhat protective against SSB. While EIPT is facilitated in solution compared to the dry environment, the above findings are consistent with the stability of DNA against hydrated electron-induced SSB in solution versus free electron-induced SSB formation in dry DNA.

Electron-Induced Decomposition of 5-Bromo-4-thiouracil and 5-Bromo-4-thio-2'-deoxyuridine: The Effect of the Deoxyribose Moiety on Dissociative Electron Attachment.

When modified uridine derivatives are incorporated into DNA, radical species may form that cause DNA damage. This category of molecules has been proposed as radiosensitizers and is currently being researched. Here, we study electron attachment to 5-bromo-4-thiouracil (BrSU), a uracil derivative, and 5-bromo-4-thio-2'-deoxyuridine (BrSdU), with an attached deoxyribose moiety via the N-glycosidic (N1-C) bond. Quadrupole mass spectrometry was used to detect the anionic products of dissociative electron attachment (DEA), and the experimental results were supported by quantum chemical calculations performed at the M062X/aug-cc-pVTZ level of theory. Experimentally, we found that BrSU predominantly captures low-energy electrons with kinetic energies near 0 eV, though the abundance of bromine anions was rather low compared to a similar experiment with bromouracil. We suggest that, for this reaction channel, proton-transfer reactions in the transient negative ions limit the release of bromine anions.

Inactive-to-Active Transition of Human Thymidine Kinase 1 Revealed by Molecular Dynamics Simulations.

Despite its importance in the nucleoside (and nucleoside prodrug) metabolism, the structure of the active conformation of human thymidine kinase 1 (hTK1) remains elusive. We perform microsecond molecular dynamics simulations of the inactive enzyme form bound to a bisubstrate inhibitor that was shown experimentally to activate another TK1-like kinase, Thermotoga maritima TK (TmTK). Our results are in excellent agreement with the experimental findings for the TmTK closed-to-open state transition. We show that the inhibitor induces an increase of the enzyme radius of gyration due to the expansion on one of the dimer interfaces; the structural changes observed, including the active site pocket volume increase and the decrease in the monomer-monomer buried surface area and of the number of hydrogen bonds (as compared to the inactive enzyme control simulation), indicate that the catalytically competent (open) conformation of hTK1 can be assumed in the presence of an activating ligand.

Extracellular Vesicle Mediated Vascular Pathology in Glioblastoma.

Glioblastoma (GBM) is an incurable, infiltrative high-grade brain tumour associated with dramatic vascular responses observed both locally (angiogenesis, vascular cooption, angiocrine effects, microthrombosis) and systemically (venous thromboembolism). GBM-associated vascular pathology is diagnostically relevant and constitutes a source of morbidity, mortality and progressive changes in tumour biology. Extracellular vesicles (EVs) have emerged as unique mediators of vascular effects in brain tumours acting as vehicles for intercellular transfer of oncoproteins (e.g. EGFRvIII), RNA, DNA and molecular effectors of angiogenesis and thrombosis. Vascular effects of GBM EVs are regulated by cancer cell genome, epigenome and microenvironment and differ between subtypes of cancer cells and stem cells. Understanding and targeting EV-driven vascular processes in GBM may offer new approaches to diagnose and treat these intractable tumours.

Nanofluidics for Simultaneous Size and Charge Profiling of Extracellular Vesicles.

Extracellular vesicles (EVs) are cell-derived membrane structures that circulate in body fluids and show considerable potential for noninvasive diagnosis. EVs possess surface chemistries and encapsulated molecular cargo that reflect the physiological state of cells from which they originate, including the presence of disease. In order to fully harness the diagnostic potential of EVs, there is a critical need for technologies that can profile large EV populations without sacrificing single EV level detail by averaging over multiple EVs. Here we use a nanofluidic device with tunable confinement to trap EVs in a free-energy landscape that modulates vesicle dynamics in a manner dependent on EV size and charge. As proof-of-principle, we perform size and charge profiling of a population of EVs extracted from human glioblastoma astrocytoma (U373) and normal human astrocytoma (NHA) cell lines.

Influence of Hypoxia on Radiosensitization of Cancer Cells by 5-Bromo-2'-deoxyuridine.

Radiotherapy is a crucial cancer treatment, but its outcome is still far from satisfactory. One of the reasons that cancer cells show resistance to ionizing radiation is hypoxia, defined as a low level of oxygenation, which is typical for solid tumors. In the hypoxic environment, cancer cells are 2-3 times more resistant to ionizing radiation than normoxic cells. To overcome this important impediment, radiosensitizers should be introduced to cancer therapy. When modified with an electrophilic substituent, nucleosides may undergo efficient dissociative electron attachment (DEA) that leaves behind nucleoside radicals, which, in secondary reactions, are able to induce DNA damage, leading to cancer cell death. We report the radiosensitizing effect of one of the best-known DEA-type radiosensitizers-5-bromo-2'-deoxyuridine (BrdU)-on breast (MCF-7) and prostate (PC3) cancer cells under both normoxia and hypoxia. MCF-7 and PC3 cells were treated with BrdU to investigate the effect of hypoxia on cell proliferation, incorporation into DNA and radiosensitivity. While the oxygen concentration did not significantly affect the efficiency of BrdU incorporation into DNA or the proliferation of tumor cells, the radiosensitizing effect of BrdU on hypoxic cells was more evident than on normoxic cells. Further mechanistic studies performed with the use of flow cytometry showed that under hypoxia, BrdU increased the level of histone H2A.X phosphorylation after X-ray exposure to a greater extent than under normal oxygenation conditions. These results confirm that the formation of double-strand breaks in hypoxic BrdU-treated cancer cells is more efficient. In addition, by performing stationary radiolysis of BrdU solution in the presence of an ●OH radical scavenger, we compared the degree of its electron-induced degradation under aerobic and anaerobic conditions. It was determined that radiodegradation under anaerobic conditions was almost twice as high as that under aerobic conditions.

Electron Attachment to 5-Fluorouracil: The Role of Hydrogen Fluoride in Dissociation Chemistry.

We investigate dissociative electron attachment to 5-fluorouracil (5-FU) employing a crossed electron-molecular beam experiment and quantum chemical calculations. Upon the formation of the 5-FU- anion, 12 different fragmentation products are observed, the most probable dissociation channel being H loss. The parent anion, 5-FU-, is not stable on the experimental timescale (~140 µs), most probably due to the low electron affinity of FU; simple HF loss and F- formation are seen only with a rather weak abundance. The initial dynamics upon electron attachment seems to be governed by hydrogen atom pre-dissociation followed by either its full dissociation or roaming in the vicinity of the molecule, recombining eventually into the HF molecule. When the HF molecule is formed, the released energy might be used for various ring cleavage reactions. Our results show that higher yields of the fluorine anion are most probably prevented through both faster dissociation of an H atom and recombination of F- with a proton to form HF. Resonance calculations indicate that F- is formed upon shape as well as core-excited resonances.

SMARCB1 loss induces druggable cyclin D1 deficiency via upregulation of MIR17HG in atypical teratoid rhabdoid tumors.

Atypical teratoid rhabdoid tumor (ATRT) is a fatal pediatric malignancy of the central neural system lacking effective treatment options. It belongs to the rhabdoid tumor family and is usually caused by biallelic inactivation of SMARCB1, encoding a key subunit of SWI/SNF chromatin remodeling complexes. Previous studies proposed that SMARCB1 loss drives rhabdoid tumor by promoting cell cycle through activating transcription of cyclin D1 while suppressing p16. However, low cyclin D1 protein expression is observed in most ATRT patient tumors. The underlying mechanism and therapeutic implication of this molecular trait remain unknown. Here, we show that SMARCB1 loss in ATRT leads to the reduction of cyclin D1 expression by upregulating MIR17HG, a microRNA (miRNA) cluster known to generate multiple miRNAs targeting CCND1. Furthermore, we find that this cyclin D1 deficiency in ATRT results in marked in vitro and in vivo sensitivity to the CDK4/6 inhibitor palbociclib as a single agent. Our study identifies a novel genetic interaction between SMARCB1 and MIR17HG in regulating cyclin D1 in ATRT and suggests a rationale to treat ATRT patients with FDA-approved CDK4/6 inhibitors. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Electrophilic Properties of 2'-Deoxyadenosine···Thymine Dimer: Photoelectron Spectroscopy and DFT Studies.

The anion radical of the 2'-deoxyadenosine···thymine (dAT•-) pair has been investigated experimentally and theoretically in the gas phase. By employing negative-ion photoelectron spectroscopy (PES), we have registered a spectrum typical for the valence-bound anion, featuring a broad peak at the electron-binding energy (EBE) between ∼1.5 and 2.2 eV with the maximum at ∼1.9 eV. The measured value of the adiabatic electron affinity (AEA) for dAT was estimated to be ∼1.1 eV. Calculations performed at the M06-2X/6-31++G(d,p) level revealed that the structure, where thymine is coordinated to the sugar of dA by two hydrogen bonds, is responsible for the observed PES signal. The AEAG and the vertical detachment energy of 0.91 and 1.68 eV, respectively, calculated for this structure reproduce the experimental values well. The role of the possible proton transfer in the stabilization of anionic radical complexes is discussed.

Electron-Induced Decomposition of Uracil-5-yl O-(N,N-dimethylsulfamate): Role of Methylation in Molecular Stability.

The incorporation of modified uracil derivatives into DNA leads to the formation of radical species that induce DNA damage. Molecules of this class have been suggested as radiosensitizers and are still under investigation. In this study, we present the results of dissociative electron attachment to uracil-5-yl O-(N,N-dimethylsulfamate) in the gas phase. We observed the formation of 10 fragment anions in the studied range of electron energies from 0-12 eV. Most of the anions were predominantly formed at the electron energy of about 0 eV. The fragmentation paths were analogous to those observed in uracil-5-yl O-sulfamate, i.e., the methylation did not affect certain bond cleavages (O-C, S-O and S-N), although relative intensities differed. The experimental results are supported by quantum chemical calculations performed at the M06-2X/aug-cc-pVTZ level of theory. Furthermore, a resonance stabilization method was used to theoretically predict the resonance positions of the fragment anions O- and CH3-.

Design, synthesis and biological evaluation of betulin-3-yl 2-amino-2-deoxy-ß-d-glycopyranosides.

Betulin is a natural pentacyclic triterpenoid, possessing a lupane-structure, with a wide range of pharmacological activities. Its weak hydrosolubility hinders the biological activity of the compound and its derivatives. To circumvent this problem, we synthesized and tested in vitro three d-glycosaminosides of betulin. The structure of betulin was modified by incorporation of 2-amino-2-deoxy-d-gluco- and -d-galactopyranosyl moieties to its C-3 position. So far betulinyl glycosides containing these amino-sugars have not been reported in the literature. The structure of the studied derivatives was confirmed by 1H and 13C NMR spectroscopy as well as mass spectrometry. The 28-O-acetylbetulin-3-yl 2-amino-2-deoxy-ß-d-glucopyranoside and betulin-3-yl 2-amino-2-deoxy-ß-d-gluco- and ß-d-galactopyranoside were tested against the human pathogenic fungi and Gram-positive and Gram-negative bacteria. Moreover, the MTT assay of their cytotoxicity was performed on the MCF-7 breast cancer cell line and on the HDFa, human dermal fibroblasts. The Ames test on mutagenic properties completed our biological assays.

The Impact of Oncogenic EGFRvIII on the Proteome of Extracellular Vesicles Released from Glioblastoma Cells.

Glioblastoma multiforme (GBM) is a highly aggressive and heterogeneous form of primary brain tumors, driven by a complex repertoire of oncogenic alterations, including the constitutively active epidermal growth factor receptor (EGFRvIII). EGFRvIII impacts both cell-intrinsic and non-cell autonomous aspects of GBM progression, including cell invasion, angiogenesis and modulation of the tumor microenvironment. This is, at least in part, attributable to the release and intercellular trafficking of extracellular vesicles (EVs), heterogeneous membrane structures containing multiple bioactive macromolecules. Here we analyzed the impact of EGFRvIII on the profile of glioma EVs using isogenic tumor cell lines, in which this oncogene exhibits a strong transforming activity. We observed that EGFRvIII expression alters the expression of EV-regulating genes (vesiculome) and EV properties, including their protein composition. Using mass spectrometry, quantitative proteomic analysis and Gene Ontology terms filters, we observed that EVs released by EGFRvIII-transformed cells were enriched for extracellular exosome and focal adhesion related proteins. Among them, we validated the association of pro-invasive proteins (CD44, BSG, CD151) with EVs of EGFRvIII expressing glioma cells, and downregulation of exosomal markers (CD81 and CD82) relative to EVs of EGFRvIII-negative cells. Nano-flow cytometry revealed that the EV output from individual glioma cell lines was highly heterogeneous, such that only a fraction of vesicles contained specific proteins (including EGFRvIII). Notably, cells expressing EGFRvIII released EVs double positive for CD44/BSG, and these proteins also colocalized in cellular filopodia. We also detected the expression of homophilic adhesion molecules and increased homologous EV uptake by EGFRvIII-positive glioma cells. These results suggest that oncogenic EGFRvIII reprograms the proteome and uptake of GBM-related EVs, a notion with considerable implications for their biological activity and properties relevant for the development of EV-based cancer biomarkers.

The Product of Matrix Metalloproteinase Cleavage of Doxorubicin Conjugate for Anticancer Drug Delivery: Calorimetric, Spectroscopic, and Molecular Dynamics Studies on Peptide-Doxorubicin Binding to DNA.

Matrix metalloproteinases (MMPs) are extracellular matrix degradation factors, promoting cancer progression. Hence, they could provide an enzyme-assisted delivery of doxorubicin (DOX) in cancer treatment. In the current study, the intercalation process of DOX and tetrapeptide-DOX, the product of the MMPs' cleavage of carrier-linked DOX, into dsDNA was investigated using stationary and time-resolved fluorescence spectroscopy, UV-Vis spectrophotometry and isothermal titration calorimetry (ITC). The molecular dynamics (MD) simulations on the same tetrapeptide-DOX…DNA and DOX…DNA systems were also performed. The undertaken studies indicate that DOX and tetrapeptide-DOX can effectively bond with dsDNA through the intercalation mode; however, tetrapeptide-DOX forms less stable complexes than free DOX. Moreover, the obtained results demonstrate that the differences in DNA affinity of both forms of DOX can be attributed to different intercalation modes. Tetrapeptide-DOX shows a preference to intercalate into DNA through the major groove, whereas DOX does it through the minor one. In summary, we can conclude that the tetrapeptide-DOX intercalation to DNA is significant and that even the lack of non-specific proteases releasing DOX from the tetrapeptide conjugate, the presence of which is suggested by the literature for the efficient release of DOX, should not prevent the cytostatic action of the anthracycline.

5-(N-Trifluoromethylcarboxy)aminouracil as a Potential DNA Radiosensitizer and Its Radiochemical Conversion into N-Uracil-5-yloxamic Acid.

Hypoxia-a hallmark of solid tumors-dramatically impairs radiotherapy, one of the most common anticancer modalities. The adverse effect of the low-oxygen state can be eliminated by the concomitant use of a hypoxic cell radiosensitizer. In the present paper, we show that 5-(N-trifluoromethylcarboxy) aminouracil (CF3CONHU) can be considered as an effective radiosensitizer of DNA damage, working under hypoxia. The title compound was synthesized in the reaction of 5-aminouracil and trifluoroacetic anhydride in trifluoroacetic acid. Then, an aqueous and deoxygenated solution of the HPLC purified compound containing tert-butanol as a hydroxyl radical scavenger was irradiated with X-rays. Radiodegradation in a 26.67 ± 0.31% yield resulted in only one major product-N-uracil-5-yloxamic acid. The mechanism that is possibly responsible for the formation of the observed radioproduct has been elucidated with the use of DFT calculations. The cytotoxic test against the PC3 prostate cancer cell line and HDFa human dermal fibroblasts confirmed the low cytotoxicity of CF3CONHU. Finally, a clonogenic assay and flow cytometric analysis of histone H2A.X phosphorylation proved the radiosensitization in vitro.

Oncogenic Regulation of Extracellular Vesicle Proteome and Heterogeneity.

Mutational and epigenetic driver events profoundly alter intercellular communication pathways in cancer. This effect includes deregulated release, molecular composition, and biological activity of extracellular vesicles (EVs), membranous cellular fragments ranging from a few microns to less than 100 nm in diameter and filled with bioactive molecular cargo (proteins, lipids, and nucleic acids). While EVs are usually classified on the basis of their physical properties and biogenetic mechanisms, recent analyses of their proteome suggest a larger than expected molecular diversity, a notion that is also supported by multicolour nano-flow cytometry and other emerging technology platforms designed to analyze single EVs. Both protein composition and EV diversity are markedly altered by oncogenic transformation, epithelial to mesenchymal transition, and differentiation of cancer stem cells. Interestingly, only a subset of EVs released from mutant cells may carry oncogenic proteins (e.g., EGFRvIII), hence, these EVs are often referred to as "oncosomes". Indeed, oncogenic transformation alters the repertoire of EV-associated proteins, increases the presence of pro-invasive cargo, and alters the composition of distinct EV populations. Molecular profiling of single EVs may reveal a more intricate effect of transforming events on the architecture of EV populations in cancer and shed new light on their biological role and diagnostic utility.

Extracellular vesicle communication pathways as regulatory targets of oncogenic transformation.

Pathogenesis of human cancers bridges intracellular oncogenic driver events and their impact on intercellular communication. Among multiple mediators of this 'pathological connectivity' the role of extracellular vesicles (EVs) and their subsets (exosomes, ectosomes, oncosomes) is of particular interest for several reasons. The release of EVs from cancer cells represents a unique mechanism of regulated expulsion of bioactive molecules, a process that also mediates cell-to-cell transfer of lipids, proteins, and nucleic acids. Biological effects of these processes have been implicated in several aspects of cancer-related pathology, including tumour growth, invasion, angiogenesis, metastasis, immunity and thrombosis. Notably, the emerging evidence suggests that oncogenic mutations may impact several aspects of EV-mediated cell-cell communication including: (i) EV release rate and protein content; (ii) molecular composition of cancer EVs; (iii) the inclusion of oncogenic and mutant macromolecules in the EV cargo; (iv) EV-mediated release of genomic DNA; (v) deregulation of mechanisms responsible for EV biogenesis (vesiculome) and (vi) mechanisms of EV uptake by cancer cells. Intriguingly, EV-mediated intercellular transfer of mutant and oncogenic molecules between subpopulations of cancer cells, their indolent counterparts and stroma may exert profound biological effects that often resemble (but are not tantamount to) oncogenic transformation, including changes in cell growth, clonogenicity and angiogenic phenotype, or cause cell stress and death. However, several biological barriers likely curtail a permanent horizontal transformation of normal cells through EV-mediated mechanisms. The ongoing analysis and targeting of EV-mediated intercellular communication pathways can be viewed as a new therapeutic paradigm in cancer, while the analysis of oncogenic cargo contained in EVs released from cancer cells into biofluids is being developed for clinical use as a biomarker and companion diagnostics. Indeed, studies are underway to further explore the multiple links between molecular causality in cancer and various aspects of cellular vesiculation.

Oncogenes and Clotting Factors: The Emerging Role of Tumor Cell Genome and Epigenome in Cancer-Associated Thrombosis.

There are emerging linkages between biological and genetic aspects of cancer progression and the mechanisms of cancer-associated thrombosis. It is argued that reciprocal influences between cancer cells, their associated vascular stroma, and the hemostatic system may shape the mechanism of coagulopathy. In this regard, glioblastoma multiforme offers a paradigm where the prevalent occurrence of local microthrombosis and peripheral venous thromboembolism can be linked to the profiles of oncogenic driver mutations and their impact on the expression of coagulation-related genes (coagulome). These relationships can be recapitulated in cellular models of glioblastoma, where the expression of tissue factor, podoplanin, and the release of procoagulant microparticles (extracellular vesicles) remains under the control of oncogenic pathways (epidermal growth factor receptor variant III, isocitrate dehydrogenase 1). These pathways define molecular subtypes of glioblastoma that express differential coagulomes. Moreover, single-cell sequencing of glioblastoma samples reveals a combinatorial rather than common profile of both subtype markers and coagulation-related genes. Based on these emerging observations, the authors suggest that cancers may operate as coagulant composites, where individual cells and their dominant populations express different procoagulant phenotypes, resulting in the net impact on the hemostatic system. They suggest that relating these mechanisms to clinical presentations of thrombosis may facilitate a more causality-based, personalized, and possibly cancer-specific thromboprophylaxis and treatment.