In the published version of this article [1], an overlap discrepancy was identified in some of the slides in Fig. (7). This error was unintentional and occurred during the manuscript preparation stage by the authors. This correction does not affect the study’s quantitative analyses, results, or conclusions. The authors apologize for this error and any inconvenience it may have caused. The original article can be found online at: https://www.eurekaselect.com/article/146623 The authors apologizes for this oversight and any inconvenience caused to the readers.

CRISPR-based tools have quickly moved from specialist techniques to routine instruments in biology and medicine, and they are now central to large-scale loss-of-function and perturbation screens. In this review, we focus on how pooled CRISPR screens are used to interrogate gene function in living cells, most often through cell fitness or simple selectable markers, and contrast this with arrayed formats that trade throughput for richer molecular readouts, such as transcriptome-wide changes. We bring together current strategies for library design, delivery, and selection and show how different Cas nucleases, including Cas9, Cas12, and Cas13, broaden the range of genome and transcriptome perturbations that can be assayed. We then discuss recent applications in drug response, viral infection, and cancer biology and consider how improvements in high-content technologies, data analysis, and emerging diagnostic uses are likely to shape the next generation of CRISPR-based screening studies.

Background:Pathogenic variations in the PCLO gene cause Pontocerebellar Hypoplasia type 3 (PCH3), an extremely rare autosomal recessive disease characterized by seizure, intellectual disability, developmental delay, and microcephaly. PCLO encodes the Piccolo protein, which plays a critical role in synaptic function and neurological disorders. To date, only one pathogenic PCLO variant associated with PCH3 has been reported in the literature. While research on PCH3 is ongoing, the rarity of the condition has limited the number of studies. Materials and Methods:A novel homozygous variant in PCLO (NM_033026: c.458TC, p. Met153Thr) was identified through wholeexome sequencing and confirmed by Sanger sequencing. Functional studies were conducted to assess the pathogenicity of this variant using next-generation sequencing (NGS), in silico analysis, CRISPR-edited cells, and real-time PCR. Results:The proband presented with seizure, microcephaly, mild ataxia, and behavioral issues. Notably, in addition to previously reported symptoms, the patient also exhibited toe-walking, loss of tendon reflexes, and unilateral paralysis. The PCLO knockout cell model and molecular analysis confirmed the loss of function of the Piccolo protein in the homozygous variant. Our findings also demonstrated that Piccolo deficiency may affect the expression of other genes, including CtBp1 and BSN. Conclusion:We identified a novel PCLO variant responsible for PCH3 in a second known family worldwide. Additionally, a CRISPR-based cell model for PCH3 was developed, providing a valuable foundation for further research into the molecular mechanisms underlying Piccolo function and disease pathogenesis.

Colorectal cancer is a common and fatal disease that affects many people globally. CRC is classified as the third most prevalent cancer among males and the second most frequent cancer among females worldwide. The purpose of this article is to examine how personalized medicine might be used to treat colorectal cancer. The classification of colorectal cancer based on molecular profiling, including the detection of significant gene mutations, genomic instability, and gene dysregulation, is the main topic of this discussion. Advanced technologies and biomarkers are among the detection methods that are explored, demonstrating their potential for early diagnosis and precise prognosis. In addition, the essay explores the world of treatment possibilities by providing light on FDA-approved personalized medicine solutions that provide individualized and precise interventions based on patient characteristics. This article assesses targeted treatments like cetuximab and nivolumab, looks at the therapeutic usefulness of biomarkers like microsatellite instability (MSI) and circulating tumor DNA (ctDNA), and investigates new approaches to combat resistance. Through this, our review provides a thorough overview of personalized medicine in the context of colorectal cancer, ultimately highlighting its potential to revolutionize the field and improve patient care.

Kartogenin (KGN) is a small synthetic heterocyclic molecule with chondrogenic and chondroprotective effects. Since its discovery, there has been a focus on regenerating cartilage damage and treating Osteoarthritis) OA(. In the treatment of OA, it’s important to target both cartilage and subchondral bone. KGN appears to reduce cartilage degradation and changes in subchondral trabecular bone. It can also reduce inflammation and pain behavior in vitro and in vivo. Additionally, KGN promotes chondrocyte differentiation and proliferation. It has been applied in many regenerative research fields including aesthetic procedures, limb skeletal growth, wound healing, tendon and bone regeneration and disc regeneration. KGN is similar to the natural ligands involved in cell signaling and differentiation. The master regulator of cartilage genes, Sex‑determining region‑box 9 protein (SOX9), is upregulated by KGN, making it an ideal drug to promote cartilage repair. Advantages of KGN include demonstrated low toxicity across various cell types and no apparent adverse effects in animals. It is highly stable, easily stored at room temperature, and can be synthesized inexpensively and efficiently. Analogues of KGN have entered clinical trials.

Background: Pathogenic variations in the PCLO gene cause Pontocerebellar Hypoplasia type 3 (PCH3), an extremely rare autosomal recessive disease characterized by seizure, intellectual disability, developmental delay, and microcephaly. PCLO encodes the Piccolo protein, which plays a critical role in synaptic function and neurological disorders. To date, only one pathogenic PCLO variant associated with PCH3 has been reported in the literature. While research on PCH3 is ongoing, the rarity of the condition has limited the number of studies. Materials and Methods: A novel homozygous variant in PCLO (NM_033026: c.458T>C, p. Met153Thr) was identified through wholeexome sequencing and confirmed by Sanger sequencing. Functional studies were conducted to assess the pathogenicity of this variant using next-generation sequencing (NGS), in silico analysis, CRISPR-edited cells, and real-time PCR. Results: The proband presented with seizure, microcephaly, mild ataxia, and behavioral issues. Notably, in addition to previously reported symptoms, the patient also exhibited toe-walking, loss of tendon reflexes, and unilateral paralysis. The PCLO knockout cell model and molecular analysis confirmed the loss of function of the Piccolo protein in the homozygous variant. Our findings also demonstrated that Piccolo deficiency may affect the expression of other genes, including CtBp1 and BSN. Conclusion: We identified a novel PCLO variant responsible for PCH3 in a second known family worldwide. Additionally, a CRIS-PR-based cell model for PCH3 was developed, providing a valuable foundation for further research into the molecular mechanisms underlying Piccolo function and disease pathogenesis.

Background Polycystic ovary syndrome (PCOS) is one of the most prevalent endocrine and metabolic disorders, affecting approximately 15% of females. This syndrome is characterized by its remarkable heterogeneity, and epigenetic factors may contribute to its development. Since the PPARG gene is crucial for lipid and glucose metabolism, this study investigated the correlation between hyperinsulinemia, hyperandrogenism, and PPARG1 methylation in granulosa cells of polycystic ovary syndrome patients. Methods Follicular fluid was collected from 30 healthy control subjects, 25 patients with PCOS, and further subdivided into groups of 20 patients with PCOS exhibiting hyperandrogenism and insulin resistance, respectively. Granulosa cells were isolated from the follicular fluid. Subsequently, RNA and DNA were extracted, and bisulfite conversion was performed to analyze DNA methylation. The methylation status of the PPARG1 gene was assessed using MS-PCR, and qRT-PCR was employed to quantify PPARG1 mRNA expression. Results Analysis of the data revealed significant differences in PPARG1 gene methylation among the four patient groups. Notably, the PPARG1 gene promoter exhibited hypermethylation in both the PCOS-IR and PCOS-HA groups. This hypermethylation was associated with subsequent downregulation of PPARG1 gene expression compared to the control group ( p < 0.05). Conclusion Our findings provide compelling evidence for the epigenetic regulation of PPARG1 in PCOS. PPARG1 promoter hypermethylation was observed specifically in PCOS patients with insulin resistance (IR) and hyperandrogenism (HA), suggesting a potential role for DNA methylation in the downregulation of PPARG1 expression in these subgroups. This link between PPARG1 methylation and gene expression required further investigation to elucidate its functional significance in the pathogenesis of PCOS.

Background: Critical limb ischemia (CLI) is considered the most severe form of peripheral artery disease (PAD). Nowadays, using stem cells such as mesenchymal stem cells (MSCs) to induce angiogenesis seems like a promising method for CLI therapy. Among the many factors that affect the angiogenesis process, microRNA-126 has an important role. Objective: The goal of this study was to increase the angiogenic potential of bone marrow mesenchymal stem cells (BMSCs) via using microRNA-126. Methods: BMSCs were isolated from male C57BL/6 inbred mice. CLI model was created by femoral artery ligation on C57BL/6 mice. Animals were allocated to control, BMSCs, miR-126, and BMSCsmiR-126 groups, and a defined number of the cells and virus were injected 24 h after surgery. Then, wound-healing assay, functional tests, real-time PCR, histopathological evaluation, and donor cell survival were performed. Results: Results showed that BMSCs and miR-126 groups had a positive effect on angiogenesis. BMSCs miR-126 group had a significant effect on functional improvements, endothelial cell migration, neovascularization, and muscle restructures. In vivo evaluation showed that miR-126 could increase BMSCs survival and paracrine secretion of angiogenic factors such as VEGF and led to remarkable functional improvements and neovascularization in ischemic tissues. Conclusion: It can be concluded that the combination uses of BMSCs and miR-126 lead to more effective recovery from ischemic damage compared with using them alone. MiR-126 can be used as a strong modifier to reinforce the angiogenic potential, paracrine secretion, and survival of the BMSCs.

The groundbreaking gene-editing tool CRISPR-Cas9 has revolutionized molecular biology. It is a method that enables scientists to precisely alter the DNA of living cells, which holds great promise for treating genetic illnesses. The Cas9 protein and a guide RNA (gRNA) are the two critical parts of the CRISPR-Cas9 system. While the gRNA guides the Cas9 protein to the appropriate region in the genome, the Cas9 protein functions as a pair of molecular scissors, cutting the DNA at a specific location. In recent years, several databases have been created to assist researchers in designing efficient gRNAs. These databases provide information on the specificity and efficiency of various gRNAs and tools for designing custom gRNAs. This review article will go over the most beneficial and recent databases for gRNA design that are now accessible. These databases are crucial for attaining effective and precise gene editing and therapy using the CRISPR-Cas9 system.

Evidence suggests that downregulation of miR-153 in hepatocellular carcinoma (HCC) cells and tissues promotes cell proliferation. One of the characteristics of cancer cells is the high expression of B-cell lymphoma 2 (BCL2) gene inside the cells, which is a barrier to cell apoptosis. In this study, cloned miR-153 transfection into the HepG2 cancer cells altered the expression of genes associated with autophagy, reduced the BCL2 gene expression, and resulted in apoptosis, as assessed by real-time PCR. The BCL2 gene expression decreased significantly, P < 0.01, in the miR-153-transfected cells, compared to the negative control and empty vector-transfected cells. This analysis was performed using real-time PCR, and its comparison was evaluated using Prism software. This reduction disrupted the Beclin 1 and BCL2 complexes as a type of signal to initiate the process. Also, the expression of light chain 3 and BECN1 genes increased as autophagic genes. The cloning of miR-153 in the PLKO1 vector was found and to be effective, and the expression of miR-153 was confirmed inside the target cells. By targeting the BCL2 gene, reducing its expression, and increasing the expression of LC3 and BECN1 genes in cancer cells, miR-153 might cause apoptosis and autophagy within the HCC cell line. The luciferase assay was used for gene targeting and results was analyzed by ANOVA.

Aging causes numerous age-related diseases, leading the human species to death. Nevertheless, rejuvenating strategies based on cell epigenetic modifications are a possible approach to counteract disease progression while getting old. Cell reprogramming of adult somatic cells toward pluripotency ought to be a promising tool for age-related diseases. However, researchers do not have control over this process as cells lose their fate, and cause potential cancerous cells or unexpected cell phenotypes. Direct and partial reprogramming were introduced in recent years with distinctive applications. Although direct reprogramming makes cells lose their identity, it has various applications in regeneration medicine. Temporary and regulated in vivo overexpression of Yamanaka factors has been shown in several experimental contexts to be achievable and is used to rejuvenate mice models. This regeneration can be accomplished by altering the epigenetic adult cell signature to the signature of a younger cell. The greatest advantage of partial reprogramming is that this method does not allow cells to lose their identity when they are resetting their epigenetic clock. It is a regimen of short-term Oct3/4, Sox2, Klf4, and c-Myc expression in vivo that prevents full reprogramming to the pluripotent state and avoids both tumorigenesis and the presence of unwanted undifferentiated cells. We know that many neurological age-related diseases, such as Alzheimer ' s disease, stroke, dementia, and Parkinson's disease, are the main cause of death in the last decades of life. Therefore, scientists have a special tendency regarding neuroregeneration methods to increase human life expectancy.

Niemann–Pick disease (NPD) is another type of metabolic disorder that is classified as lysosomal storage diseases (LSDs). The main cause of the disease is mutation in the SMPD1 (type A and B) or NPC1 or NPC2 (type C) genes, which lead to the accumulation of lipid substrates in the lysosomes of the liver, brain, spleen, lung, and bone marrow cells. This is followed by multiple cell damage, dysfunction of lysosomes, and finally dysfunction of body organs. So far, about 346, 575, and 30 mutations have been reported in SMPD1 , NPC1 , and NPC2 genes, respectively. Depending on the type of mutation and the clinical symptoms of the disease, the treatment will be different. The general aim of the current study is to review the clinical and molecular characteristics of patients with NPD and study various treatment methods for this disease with a focus on gene therapy approaches.

Background FCSK-congenital disorder of glycosylation (FCSK-CDG) is a recently discovered rare autosomal recessive genetic disorder with defective fucosylation due to mutations in the fucokinase encoding gene, FCSK. Despite the essential role of fucokinase in the fucose salvage pathway and severe multisystem manifestations of FCSK-CDG patients, it is not elucidated which cells or which types of fucosylation are affected by its deficiency. Methods In this study, CRISPR/Cas9 was employed to construct an FCSK-CDG cell model and explore the molecular mechanisms of the disease by lectin flow cytometry and real-time PCR analyses. Results Comparison of cellular fucosylation by lectin flow cytometry in the created CRISPR/Cas9 FCSK knockout and the same unedited cell lines showed no significant change in the amount of cell surface fucosylated glycans, which is consistent with the only documented previous study on different cell types. It suggests a probable effect of this disease on secretory glycoproteins. Investigating O-fucosylation by analysis of the NOTCH3 gene expression as a potential target revealed a significant decrease in the FCSK knockout cells compared with the same unedited ones, proving the effect of fucokinase deficiency on EGF-like repeats O-fucosylation. Conclusion This study expands insight into the FCSK-CDG molecular mechanism; to the best of our knowledge, it is the first research conducted to reveal a gene whose expression level alters due to this disease.

GM1 gangliosidosis is one type of hereditary error of metabolism that occurs due to the absence or reduction of β-galactosidase enzyme content in the lysosome of cells, including neurons. In vitro, the use of neural cell lines could facilitate the study of this disease. By creating a cell model of GM1 gangliosidosis on the SH-SY5Y human nerve cell line, it is possible to understand the main role of this enzyme in breaking down lipid substrate and other pathophysiologic phenomena this disease. To knock-out the human GLB1 gene, guides targeting exons 14 and 16 of the GLB1 gene were designed using the CRISPOR and CHOP-CHOP websites, and high-efficiency guides were selected for cloning in the PX458 vector. After confirming the cloning, the vectors were transformed into DH5α bacteria and then the target vector was extracted and transfected into human nerve cells (SH-SY5Y cell line) by electroporation. After 48 h, GFP+ cells were sorted using the FACS technique and homozygous (compound heterozygous) single cells were isolated using the serial dilution method and sequencing was done to confirm them. Finally, gap PCR tests, X-gal and Periodic acid-Schiff (PAS) staining, and qPCR were used to confirm the knock-out of the human GLB1 gene. Additionally, RNA sequencing data analysis from existing data of the Gene Expression Omnibus (GEO) was used to find the correlation of GLB1 with other genes, and then the top correlated genes were tested for further evaluation of knock-out effects. The nonviral introduction of two guides targeting exons 14 and 16 of the GLB1 gene into SH-SY5Y cells led to the deletion of a large fragment with a size of 4.62 kb. In contrast to the non-transfected cell, X-gal staining resulted in no blue color in GLB1 gene knock-out cells indicating the absence of β-galactosidase enzyme activity in these cells. Real-time PCR (qPCR) results confirmed the RNA-Seq analysis outcomes on the GEO data set and following the GLB1 gene knock-out, the expression of its downstream genes, NEU1 and CTSA, has been decreased. It has been also shown that the downregulation of GLB1-NEU1-CTSA complex gene was involved in suppressed proliferation and invasion ability of knock-out cells. This study proved that using dual guide RNA can be used as a simple and efficient tool for targeting the GLB1 gene in nerve cells and the knockout SH-SY5Y cells can be used as a model investigation of basic and therapeutic surveys for GM1 gangliosidosis disease.

The link between type 2 diabetes mellitus (T2DM) and an increased risk of breast cancer (BC) has prompted the exploration of novel therapeutic strategies targeting shared metabolic pathways. This review focuses on the emerging evidence surrounding the potential anti-cancer effects of sodium-glucose cotransporter-2 (SGLT2) inhibitors in the context of BC. Preclinical studies have demonstrated that various SGLT2 inhibitors, such as canagliflozin, dapagliflozin, ipragliflozin, and empagliflozin, can inhibit the proliferation of BC cells, induce apoptosis, and modulate key cellular signaling pathways. These mechanisms include the activation of AMP-activated protein kinase (AMPK), suppression of mammalian target of rapamycin (mTOR) signaling, and regulation of lipid metabolism and inflammatory mediators. The combination of SGLT2 inhibitors with conventional treatments, including chemotherapy and radiotherapy, as well as targeted therapies like phosphoinositide 3-kinases (PI3K) inhibitors, has shown promising results in enhancing the anti-cancer efficacy and potentially reducing treatment-related toxicities. The identification of specific biomarkers or genetic signatures that predict responsiveness to SGLT2 inhibitor therapy could enable more personalized treatment selection and optimization, particularly for challenging BC subtypes [e, g., triple negative BC (TNBC)]. Ongoing and future clinical trials investigating the use of SGLT2 inhibitors, both as monotherapy and in combination with other agents, will be crucial in elucidating their translational potential and guiding their integration into comprehensive BC care. Overall, SGLT2 inhibitors represent a novel and promising therapeutic approach with the potential to improve clinical outcomes for patients with various subtypes of BC, including the aggressive and chemo-resistant TNBC.

BACKGROUND:Virtual screening (VS) is essential for analyzing potential drug candidates in drug discovery. Often, this involves the conversion of large volumes of compound data into specific formats suitable for computational analysis. Managing and processing this wealth of information, especially when dealing with vast numbers of compounds in various forms, such as names, identifiers, or SMILES strings, can present significant logistical and technical challenges. METHODS:To streamline this process, we developed PyComp, a software tool using Python's PyQt5 library, and compiled it into an executable with Pyinstaller. PyComp provides a systematic way for users to retrieve and convert a list of compound names, IDs (even in a range), or SMILES strings into the desired 3D format. RESULTS:PyComp greatly enhances the efficiency of data extraction, conversion, and storage processes involved in VS. It searches for similar compounds coupled with its ability to handle misidentified compounds and offers users an easy-to-use, customizable tool for managing largescale compound data. By streamlining these operations, PyComp allows researchers to save significant time and effort, thus accelerating the pace of drug discovery research. CONCLUSION:PyComp effectively addresses some of the most pressing challenges in highthroughput VS: efficient management and conversion of large volumes of compound data. As a user-friendly, customizable software tool, PyComp is pivotal in improving the efficiency and success of large-scale drug screening efforts, paving the way for faster discovery of potential therapeutic compounds.

Breast cancer (BC) is viewed as a significant public health issue and is the primary cause of cancer-related deaths among women worldwide. Triple-negative breast cancer (TNBC) is a particularly aggressive subtype that predominantly affects young premenopausal women. The tumor suppressor p53 playsa vital role in the cellular response to DNA damage, and its loss or mutations are commonly present in many cancers, including BC. Recent evidence suggests that mutant p53 proteins can aggregate and form prion-like structures, which may contribute to the pathogenesis of different types of malignancies, such as BC. This review provides an overview of BC molecular subtypes, the epidemiology of TNBC, and the role of p53 in BC development. We also discuss the potential implications of prion-like aggregation in BC and highlight future research directions. Moreover, a comprehensive analysis of the current therapeutic approaches targeting p53 aggregates in BC treatment is presented. Strategies including small molecules, chaperone inhibitors, immunotherapy, CRISPR-Cas9, and siRNA are discussed, along with their potential benefits and drawbacks. The use of these approaches to inhibit p53 aggregation and degradation represents a promising target for cancer therapy. Future investigations into the efficacy of these approaches against various p53 mutations or binding to non-p53 proteins should be conducted to develop more effective and personalized therapies for BC treatment.

Background: A rare heterozygous DYRK1B mutation (R102C) recently linked to a familial form of metabolic syndrome prompted this study to introduce the R102C mutation into the mouse DYRK1B gene, utilizing recombinant lentiviruses for long-term gene expression. Methods: In the present fundamental study, the DYRK1B R102C mutation was generated via Overlap Extension-PCR (OE-PCR) and inserted into the LeGO-iG2 transfer vector with a GFP marker. Recombinant lentiviruses were produced by co-transfection of the transfer vector carrying DYRK1B R102C, psPAX2 (Packaging vector), and pMD2 (Envelope vector) into HEK-293T cells. Results: The accuracy of the intended mutation was confirmed through OE-PCR and sequencing. Expression of DYRK1B and successful gene transfer were visualized using a fluorescence microscope to detect the GFP marker. Lentiviral titer was quantified using flow cytometry, with an infection efficiency of 108 TU/ml in HEK-293T cells. Conclusion: DYRK1B plays a crucial role in the pathogenesis of metabolic syndrome, central obesity, early-onset coronary artery disease, hypertension, type 2 diabetes, and adipogenesis, suggesting its potential as a target for therapeutic interventions. Lentiviruses carrying the DYRK1B R102C mutation offer significant advantages for both in vitro and in vivo research on metabolic syndrome. This study showcases the successful application of recombinant lentiviral vectors for gene transfer into eukaryotic cells.

Breast cancer, characterized by genetic diversity and molecular subtypes, presents significant treatment challenges, especially in human epidermal growth factor receptor type 2 (HER2)-positive cases, which are associated with poor prognosis. Metformin, widely known for its antidiabetic effects, has emerged as a promising candidate for cancer therapy. This study investigates the effect of metformin on miR-125a promoter methylation and its subsequent impact on the HER2 signaling pathway in HER2-positive breast cancer cells (SK-BR3). SK-BR3 cells were cultured and treated with various concentrations of metformin to assess its effects on cell viability, DNA methylation, HER2, and DNA Methyltransferase 1 (DNMT1) expression. Molecular analyses focus on the miR-125a signaling pathway modulation, DNA methylation, mRNA expression of DNMT1, and protein level of HER2. Research showed a dose-dependent reduction in cell viability, with IC50 values from 65 mM at 48 hours to 35 mM at 72 hours. Metformin treatment led to demethylation of the miR-125a promoter, which increased miR-125a expression and subsequently reduced HER2 levels. This suggests that metformin exerts its anticancer effects partly by regulation of the miR-125a-HER2 axis. Additionally, metformin inhibited vimentin expression, indicating its potential to interfere with epithelial-mesenchymal transition (EMT) processes. Metformin may serve as a targeted therapeutic agent in HER2-positive breast cancer by modulating the miR-125a-HER2 axis and influencing on the epigenetic and EMT regulation. Further research is warranted to elucidate the therapeutic potential of metformin through these mechanisms.

Tissue factor (TF) is an integral transmembrane protein associated with the extrinsic coagulation pathway. TF gene expression is regulated in response to inflammatory cytokines, bacterial lipopolysaccharides, and mechanical injuries. TF activity may be affected by phosphorylation of its cytoplasmic domain and alternative splicing. TF acts as the primary initiator of physiological hemostasis, which prevents local bleeding at the injury site. However, aberrant expression of TF, accompanied by the severity of diseases and infections under various pathological conditions, triggers multiple signaling pathways that support thrombosis, angiogenesis, inflammation, and metastasis. Protease-activated receptors (PARs) are central in the downstream signaling pathways of TF. In this study, we have reviewed the TF signaling pathways in different pathological conditions, such as wound injury, asthma, cardiovascular diseases (CVDs), viral infections, cancer and pathological angiogenesis. Angiogenic activities of TF are critical in the repair of wound injuries and aggressive behavior of tumors, which are mainly performed by the actions of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1 (HIF1-α). Pro-inflammatory effects of TF have been reported in asthma, CVDs and viral infections, including COVID-19, which result in tissue hypertrophy, inflammation, and thrombosis. TF-FVII induces angiogenesis via clotting-dependent and -independent mechanisms. Clottingdependent angiogenesis is induced via the generation of thrombin and cross-linked fibrin network, which facilitate vessel infiltration and also act as a reservoir for endothelial cells (ECs) growth factors. Expression of TF in tumor cells and ECs triggers clotting-independent angiogenesis through induction of VEGF, urokinase-type plasminogen activator (uPAR), early growth response 1 (EGR1), IL8, and cysteine-rich angiogenic inducer 61 (Cyr61).