Synthetic glycolipids for glioma growth inhibition developed from neurostatin and NF115 compound

Neurostatin, a natural glycosphingolipid, and NF115, a synthetic glycolipid, are inhibitors of glioma growth. While neurostatin shows high inhibitory activity on gliomas its abundance is low in mammalian brain. On the contrary NF115 exhibits less inhibitory activity on gliomas, but could be prepared by chemical synthesis. In this study we describe synthetic compounds, structurally related to NF115, capable of inhibiting glioma growth at low micromolar range. We used DNA microarray technology to compare the genes inhibited in U373-MG human glioma cells after treatment with the natural or synthetic inhibitor. New synthetic compounds were developed to interact with the product of Rho GDP dissociation inhibitor alpha gene, which was repressed in both treatments. Compounds that were inhibitors of glioma cell growth in assays for [3H]-thymidine incorporation were then injected in C6 tumor bearing rats and the tumor size in each animal group were measured. The GC-17, GC-4 and IG-5 are new compounds derived from NF115 and showed high antiproliferative activity on tumor cell lines. The GC-17 compound inhibited U373-MG glioblastoma cells (3.2 μM), the effects was fifty times more potent than NF115, and caused a significant reduction of tumor volume (P < 0.05) when tested in Wistar rats allotransplanted with C6 glioma cells.     

Selection of DNA Aptamers against Glioblastoma Cells with High Affinity and Specificity

Background

Glioblastoma is the most common and most lethal form of brain tumor in human. Unfortunately, there is still no effective therapy to this fatal disease and the median survival is generally less than one year from the time of diagnosis. Discovery of ligands that can bind specifically to this type of tumor cells will be of great significance to develop early molecular imaging, targeted delivery and guided surgery methods to battle this type of brain tumor.

Methodology/Principal Findings

We discovered two target-specific aptamers named GBM128 and GBM131 against cultured human glioblastoma cell line U118-MG after 30 rounds selection by a method called cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX). These two aptamers have high affinity and specificity against target glioblastoma cells. They neither recognize normal astraglial cells, nor do they recognize other normal and cancer cell lines tested. Clinical tissues were also tested and the results showed that these two aptamers can bind to different clinical glioma tissues but not normal brain tissues. More importantly, binding affinity and selectivity of these two aptamers were retained in complicated biological environment.

Conclusion/Significance

The selected aptamers could be used to identify specific glioblastoma biomarkers. Methods of molecular imaging, targeted drug delivery, ligand guided surgery can be further developed based on these ligands for early detection, targeted therapy, and guided surgery of glioblastoma leading to effective treatment of glioblastoma.     

Cofactor requirement for human immunodeficiency virus type 1 entry into a CD4-expressing human cell line.

Expression of the human immunodeficiency virus type 1 (HIV-1) receptor CD4 on many nonhuman and some human cell lines is not sufficient to permit HIV-1 infection. We describe a human glioblastoma cell line (U373-MG) which remains resistant to HIV-1 despite the added expression of an authentic CD4 molecule. The block to HIV-1 infection of these cells is strain independent and appears to be at viral entry. Heterokaryons of CD4-expressing U373-MG (U373-CD4) cells fused to HeLa cells allow HIV-1 entry. A U373-CD4/HeLa hybrid clone allows efficient HIV-1 replication. These results suggest that HeLa cells express a factor(s) that can complement the viral entry defect of U373-CD4 cells and is necessary for efficient CD4-mediated HIV-1 infection.     

Phosphatidylinositol 3-Kinase/AKT Pathway Inhibition by Doxazosin Promotes Glioblastoma Cells Death,Upregulation of p53 and Triggers Low Neurotoxicity

Glioblastoma is the most frequent and malignant brain tumor. Treatment includes chemotherapy with temozolomide concomitant with surgical resection and/or irradiation. However, a number of cases are resistant to temozolomide, as well as the human glioblastoma cell line U138-MG. We investigated doxazosin’s (an antihypertensive drug) activity against glioblastoma cells (C6 and U138-MG) and its neurotoxicity on primary astrocytes and organoptypic hippocampal cultures. For this study, the following methods were used: citotoxicity assays, flow cytometry, western-blotting and confocal microscopy. We showed that doxazosin induces cell death on C6 and U138-MG cells. We observed that doxazosin’s effects on the PI3K/Akt pathway were similar as LY294002 (PI3K specific inhibitor). In glioblastoma cells treated with doxasozin, Akt levels were greatly reduced. Upon examination of activities of proteins downstream of Akt we observed upregulation of GSK-3β and p53. This led to cell proliferation inhibition, cell death induction via caspase-3 activation and cell cycle arrest at G0/G1 phase in glioblastoma cells. We used in this study Lapatinib, a tyrosine kinase inhibitor, as a comparison with doxazosin because they present similar chemical structure. We also tested the neurocitotoxicity of doxazosin in primary astrocytes and organotypic cultures and observed that doxazosin induced cell death on a small percentage of non-tumor cells. Aggressiveness of glioblastoma tumors and dismal prognosis require development of new treatment agents. This includes less toxic drugs, more selective towards tumor cells, causing less damage to the patient. Therefore, our results confirm the potential of doxazosin as an attractive therapeutic antiglioma agent.     

Genomic organization, chromosomal localization and regulation of expression of the neuronal nuclear matrix protein NRP/B in human brain tumors

The nuclear matrix and its role in cell physiology are largely unknown, and the discovery of any matrix constituent whose expression is tissue- and/or cell-specific offers a new avenue of exploration. Studies of the novel neuronal nuclear matrix protein, NRP/B, reveal that it is an early and highly specific marker of neuronal induction and development in vertebrates, since its expression is restricted mainly to the developing and mature nervous system. These studies also show that NRP/B is involved in neuronal differentiation. To further examine the structure-function of NRP/B, we have cloned and characterized the murine Nrp/b gene. The murine gene consists of four exons interrupted by three introns that span 7.6kb of DNA. The complete open reading frame is localized in exon 3, suggesting that NRP/B is highly conserved during evolution. Chromosomal analysis shows that NRP/B is localized to chromosome 13 in mouse and chromosome 5q12-13 in human.Since our previous studies demonstrated that NRP/B is expressed in primary hippocampal neurons but not in primary astrocytes, we have characterized NRP/B mRNA and protein expression in various brain cell lines and in human brain tumors. Abundant expression of NRP/B mRNA and protein was observed in human neuroblastoma cell lines (IMR32, SKN-MC, SKN-SH), in glioblastoma cell lines (A172, T98G, U87-MG, U118-MG, U138-MG, and U373-MG), in neuroglioma (H4) and astrocytoma cell lines (CCF-STTG1 and SW1088). Confocal analysis of NRP/B in U87-MG glioblastoma cells indicated nuclear localization of NRP/B. NRP/B expression was also observed in human primary brain tumors including glioblastoma multiformae and astrocytomas (total of five cases). These results suggest that NRP/B expression is upregulated in human brain tumors including glioblastomas and astrocytomas, while under normal conditions NRP/B expression is restricted to neurons. This study implicates a role for NRP/B in brain tumor development.     

Gastrin inhibits motility,decreases cell death levels and increases proliferation in human glioblastoma cell lines

Whether they are of low or high histopathological grade, human astrocytic tumors are characterized by a marked propensity to diffuse into large areas of normal brain parenchyma. This invasion relates mainly to cell motility, which enables individual cell migration to take place. The present study characterizes in vitro the gastrin-mediated effects on both the growth (cell proliferation vs. cell death) and motility dynamics of the human U87 and U373 glioblastoma cell lines. A computer-assisted phase-contrast microscope was used to track the number of mitoses versus cell deaths every 4 min over a 72-h period and so to quantitatively describe the trajectories of living U373 and U87 cells growing on plastic supports in culture media both with and without the addition of 0.1, 5, or 100 nM gastrin. While 5 or 100 nM gastrin only weakly (p < .05 to p < .01) increased cell proliferation in the U87 cell line and not in U373 one, it very significantly (p < .001) inhibited the amount of cell death at 5 and 100 nM in both the U87 and U373 lines. In addition, 5 nM gastrin markedly inhibited cell mobility in U87 (p < .00001) and U373 (p < .0001) glioblastoma models. All these data strongly suggest that gastrin plays a major role in the biological behavior of the in vitro U87 and U373 human glioblastoma cell lines in matters concerning their levels of cell motility and growth dynamics. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 373–382, 1998     

Impact of atorvastatin loaded exosome as an anti-glioblastoma carrier to induce apoptosis of U87 cancer cells in 3D culture model

Exosomes (EXOs) are naturally occurring nanosized lipid bilayers that can be efficiently used as a drug delivery system to carry small pharmaceutical, biological molecules and pass major biological barriers such as the blood-brain barrier. It was hypothesized that EXOs derived from human endometrial stem cells (hEnSCs-EXOs) can be utilized as a drug carrier to enhance tumor-targeting drugs, especially for those have low solubility and limited oral bioactivity. In this study, atorvastatin (Ato) loaded EXOs (AtoEXOs) was prepared and characterized for its physical and biological activities in tumor growth suppression of 3 D glioblastoma model. The AtoEXOs were obtained in different methods to maximize drug encapsulation efficacy. The characterization of AtoEXOs was performed for its size, stability, drug release, and in vitro anti-tumor efficacy evaluated comprising inhibition of proliferation, apoptosis induction of tumor cells. Expression of apoptotic genes by Real time PCR, Annexin V/PI, tunnel assay was studied after 72 h exposing U87 cells where encapsulated in matrigel in different concentrations of AtoEXOs (5, 10 μM). The results showed that the prepared AtoEXOs possessed diameter ranging from 30–150 nm, satisfying stability and sustainable Ato release rate. The AtoEXOs was up taken by U87 and generated significant apoptotic effects while this inhibited tumor growth of U87 cells. Altogether, produced AtoEXOs formulation due to its therapeutic efficacy has the potential to be an adaptable approach to treat glioblastoma brain tumors.     

Liposome Mediated Transfer of Marker and Cytokine Genes Into Rat and Human Glioblastoma Cells in Vitro and in Vivo

Abstract

Gene transfer of Lac Z and TNF-α gene was performed in several glioblastoma cell lines in vitro (A172, F98, U373-MG, HS683, U343-MG, N31, N64, N28, N39 and N65). In comparison to the calcium phosphate precipitation technique (CPPT) the cationic liposomes were more successful measured by marker gene transfer (0.76 or 2.0%, depending on the cell line). In contrast, the amount of released human TNF-α was independed from the chosen transfer technique. Since the vector pWG29del3-hTNF contains a hormone response element it was possible to stimulate the hTNF secretion by dexamethasone up to 18 fold from a basic level of 1.1 ng/ml/24h/5×10⁵ cells. A liposomal mediated marker gene transfer (Lac Z) into a rat glio-blastoma in vivo revealed a three layer area of transfected cells around the stereotactical injection channel.     

A Cy5.5-labeled phage-displayed peptide probe for near-infrared fluorescence imaging of tumor vasculature in living mice

Near-infrared (NIR) fluorescence optical imaging is an emerging imaging technique for studying diseases at the molecular level. Optical imaging with a NIR emitting fluorophore for targeting tumor vasculature offers a noninvasive method for early detection of tumor angiogenesis and efficient monitoring of response to anti-tumor vasculature therapy. The previous in vitro results demonstrated that the GX1 peptide, identified by phage-display technology, is a tumor vasculature endothelium-specific ligand. In this report, Cy5.5-conjugated GX1 peptide was evaluated in a subcutaneous U87MG glioblastoma xenograft model to investigate tumor-targeting efficacy. The in vitro flow cytometry results revealed dose-dependent binding of Cy5.5-GX1 peptide to U87MG glioma cells. In vivo optical imaging with the Cy5.5-GX1 probe exhibited rapid U87MG tumor targeting at 0.5 h p.i., and high tumor-to-background contrast at 4 h p.i. Tumor specificity of Cy5.5-GX1 was confirmed by effective blocking of tumor uptake in the presence of unlabeled GX1 peptide (20 mg/kg). Ex vivo imaging further confirmed in vivo imaging findings, and demonstrated that Cy5.5-GX1 has a tumor-to-muscle ratio (15.21 ± 0.84) at 24 h p.i. for the non-blocked group and significantly decreased ratio (6.95 ± 0.75) for the blocked group. In conclusion, our studies suggest that Cy5.5-GX1 is a promising molecular probe for optical imaging of tumor vasculature.     

The effects of cytomegalovirus on human immunodeficiency virus replication in brain-derived cells correlate with permissiveness of the cells for each virus.

Human cytomegalovirus (HCMV) is commonly found in the brains of patients with AIDS and in some cases can be detected in the same cells as can human immunodeficiency virus type 1 (HIV-1). In this study, we analyzed the patterns of replication of HIV-1 and HCMV in singly infected cells and the effects of dual infection in human brain-derived cell lines of three different origins: neuroblastoma cell lines SK-N-MC and SY5Y; astrocytoma/glioblastoma cell lines U373-MG and Hs 683; and undifferentiated glioblastoma cell lines A172 and T98G. To bypass the restriction at the adsorption/penetration step in these CD4-negative cells, we used HIV-1 (amphotropic retrovirus) pseudotypes. These HIV-1 pseudotypes infected the majority of the cells in the cultures and expressed high levels of HIV-1 gene products in all except the SY5Y cells. The cell lines differed in the ability to support HCMV infection, but coinfection with HIV-1 had no effect on HCMV replication. The A172 cells were completely nonpermissive for HCMV gene expression, while HCMV replication in the singly infected T98G and SK-N-MC cell lines was restricted at the level of some early gene products. This resulted in complete and partial inhibition, respectively, of viral DNA synthesis. Dual infection of the A172, T98G, and SK-N-MC cells had no effect on HIV-1 replication. The other three cell lines, U373-MG, Hs 683, and SY5Y, were fully permissive for HCMV replication. In the U373-MG and Hs 683 cells, HCMV markedly inhibited the synthesis of HIV-1 gene products. In contrast, a transient stimulation of HIV-1 production followed by a repression was observed in the dually infected SY5Y cells. We conclude from these results that under conditions in which both HIV-1 and HCMV can undergo fully permissive infection, HCMV can repress HIV-1 gene expression. In cells in which HCMV replication is limited but HIV-1 replicates well, there is no effect on HIV-1 gene expression. However, activation of HIV-1, at least transiently, may occur in cells in which HIV-1 gene expression is limited. These studies suggest that a threshold level of some HIV-1 gene product(s) may obscure activation or promote repression of HIV replication by HCMV.     

Anticancer activity of synthetic analogues of the phorboxazoles

The phorboxazoles are recently described natural products that are extremely cytostatic towards the National Cancer Institute's panel of 60 tumor cell lines. We report here the results of preliminary structure-activity studies of synthetic analogues of the phorboxazoles against BT-20 breast cancer, NALM-6 B-lineage ALL, U373 brain tumor, and U373 glioblastoma cell lines. These data indicate the importance of several of the natural products' structural moieties for potent anticancer activity.     

Small heat shock protein alphaB-crystallin is part of cell cycle-dependent Golgi reorganization

AlphaB-crystallin is a developmentally regulated small heat shock protein known for its binding to a variety of denatured polypeptides and suppression of protein aggregation in vitro. Elevated levels of alphaB-crystallin are known to be associated with a number of neurodegenerative pathologies such as Alzheimer disease and multiple sclerosis. Mutations in alphaB-crystallin gene have been linked to desmin related cardiomyopathy and cataractogenesis. The physiological function of this protein, however, is unknown. Using discontinuous sucrose density gradient fractionation of post-nuclear supernatants, prepared from rat tissues and human glioblastoma cell line U373MG, we have identified discrete membrane-bound fractions of alphaB-crystallin, which co-sediment with the Golgi matrix protein, GM130. Confocal microscopy reveals co-localization of alphaB-crystallin with BODIPY TR ceramide and the Golgi matrix protein, GM130, in the perinuclear Golgi in human glioblastoma U373MG cells. Examination of synchronized cultures indicated that alphaB-crystallin follows disassembly of the Golgi at prometaphase and its reassembly at the completion of cytokinesis, suggesting that this small heat shock protein, with its chaperone-like activity, may have an important role in the Golgi reorganization during cell division.     

Multifaceted role of galectin-3 on human glioblastoma cell motility

Astrocytic tumors' aggressiveness results from an imbalance between cell proliferation and cell death favoring growth, but also from the propensity of tumor cells to detach from the primary tumor site, migrate, and invade the surrounding parenchyma. Astrocytic tumor progression is known to be associated with an increased expression of galectin-3. We investigated in cell culture how galectin-3 expression affects astrocytoma cell motility. Galectin-3 deficient cells were obtained by stable transfection of the U373 glioblastoma cell line with a specific expression antisense plasmid. Cultured galectin-3 deficient glioblastoma cells showed increased motility potential on laminin and modifications in the cytoskeleton reorganization. In addition, c-DNA microarrays and quantitative immunofluorescence analysis showed that galectin-3 deficient U373 cells have an increased expression of integrins-alpha6 and -beta1, proteins known to be implicated in the regulation of cell adhesion.     

CD4-independent infection of human neural cells by human immunodeficiency virus type 1.

A number of studies have indicated that central nervous system-derived cells can be infected with human immunodeficiency virus type 1 (HIV-1). To determine whether CD4, the receptor for HIV-1 in lymphoid cells, was responsible for infection of neural cells, we characterized infectable human central nervous system tumor lines and primary fetal neural cells and did not detect either CD4 protein or mRNA. We then attempted to block infection with anti-CD4 antibodies known to block infection of lymphoid cells; we noted no effect on any of these cultured cells. The results indicate that CD4 is not the receptor for HIV-1 infection of the glioblastoma line U373-MG, medulloblastoma line MED 217, or primary human fetal neural cells.     

The immunoglobulin‐like cell adhesion molecule hepaCAM induces differentiation of human glioblastoma U373‐MG cells

Subsequent to our identification of a novel immunoglobulin‐like cell adhesion molecule hepaCAM, we showed that hepaCAM is frequently lost in diverse human cancers and is capable of modulating cell motility and growth when re‐expressed. Very recently, a molecule identical to hepaCAM (designated as GlialCAM) was found highly expressed in glial cells of the brain. Here, we demonstrate that hepaCAM is capable of inducing differentiation of the human glioblastoma U373‐MG cells. Expression of hepaCAM resulted in a significant increase in the astrocyte differentiation marker glial fibrillary acid protein (GFAP), indicating that hepaCAM promotes glioblastoma cells to undergo differentiation. To determine the relationship between hepaCAM expression level and cell differentiation, we established two U373‐MG cell lines expressing hepaCAM at different levels. The results revealed that high‐level hepaCAM triggered a clear increase in GFAP expression as well as morphological changes characteristic of glioblastoma cell differentiation. Furthermore, high expression of hepaCAM significantly accelerated cell adhesion but inhibited cell proliferation and migration. Concomitantly, deregulation of cell cycle regulatory proteins was detected. Expectedly, the differentiation was noticeably less apparent in cells expressing low‐level hepaCAM. Taken together, our findings suggest that hepaCAM induces differentiation of the glioblastoma U373‐MG cells. The degree of cell differentiation is dependent on the expression level of hepaCAM. J. Cell. Biochem. 107: 1129–1138, 2009. © 2009 Wiley‐Liss, Inc.     

Development of Spray Dried Liposomal Dry Powder Inhaler of Dapsone

This investigation was undertaken to evaluate practical feasibility of site specific pulmonary delivery of liposomal encapsulated Dapsone (DS) dry powder inhaler for prolonged drug retention in lungs as an effective alternative in prevention of Pneumocystis carinii pneumonia (PCP) associated with immunocompromised patients. DS encapsulated liposomes were prepared by thin film evaporation technique and resultant liposomal dispersion was passed through high pressure homogenizer. DS nano-liposomes (NLs) were separated by ultra centrifugation and characterized. NLs were dispersed in phosphate buffer saline (PBS) pH 7.4 containing different carriers like lactose, sucrose, and hydrolyzed gelatin, and 15% l-leucine as antiadherent. The resultant dispersion was spray dried and spray dried formulation were characterized to ascertain its performance. In vitro pulmonary deposition was assessed using Andersen Cascade Impactor as per USP. NLs were found to have average size of 137 ± 15 nm, 95.17 ± 3.43% drug entrapment, and zeta potential of 0.8314 ± 0.0827 mV. Hydrolyzed gelatin based formulation was found to have low density, good flowability, particle size of 7.9 ± 1.1 μm, maximum fine particle fraction (FPF) of 75.6 ± 1.6%, mean mass aerodynamic diameter (MMAD) 2.2 ± 0.1 μm, and geometric standard deviation (GSD) 2.3 ± 0.1. Developed formulations were found to have in vitro prolonged drug release up to 16 h, and obeys Higuchi's Controlled Release model. The investigation provides a practical approach for direct delivery of DS encapsulated in NLs for site specific controlled and prolonged release behavior at the site of action and hence, may play a promising role in prevention of PCP.     

Tumor Necrosis Factor-α and Basic Fibroblast Growth Factor Decrease Glial Fibrillary Acidic Protein and Its Encoding mRNA in Astrocyte Cultures and Glioblastoma Cells

Abstract: Tumor necrosis factor-α is a pluripotent cytokine that is reportedly mitogenic to astrocytes. We examined expression of the astrocyte intermediate filament component glial fibrillary acidic protein in astrocyte cultures and the U373 glioblastoma cell line after treatment with tumor necrosis factor-α. Treatment with tumor necrosis factor-α for 72 h resulted in a decrease in content of glial fibrillary acidic protein and its encoding mRNA. At the same time, tumor necrosis factor-α treatment increased the expression of the cytokine interleukin-6 by astrocytes. The decrease in glial fibrillary acidic protein expression was greater when cells were subconfluent than when they were confluent. Thymidine uptake studies demonstrated that U373 cells proliferated in response to tumor necrosis factor-α, but primary neonatal astrocytes did not. However, in both U373 cells and primary astrocytes tumor necrosis factor-α induced an increase in total cellular protein content. Treatment of astrocytes and U373 cells for 72 h with the mitogenic cytokine basic fibroblast growth factor also induced a decrease in glial fibrillary acidic protein content and an increase in total protein level, demonstrating that this effect is not specific for tumor necrosis factor-α. The decrease in content of glial fibrillary acidic protein detected after tumor necrosis factor-α treatment is most likely due to dilution by other proteins that are synthesized rapidly in response to cytokine stimulation.     

Malignant human gliomas express an amiloride-sensitive Na+ conductance

Human astrocytoma cells were studied using whole cellpatch-clamp recording. An inward, amiloride-sensitiveNa⁺ current was identified in fourcontinuous cell lines originally derived from human glioblastoma cells(CH235, CRT, SKMG-1, and U251-MG) and in three primary cultures ofcells obtained from glioblastoma multiforme tumors (up to 4 passages).In addition, cells freshly isolated from a resected medulloblastomatumor displayed this same characteristic inward current. In contrast,amiloride-sensitive currents were not observed in normal humanastrocytes, low-grade astrocytomas, or juvenile pilocytic astrocytomas.The only amiloride-sensitive Na⁺channels thus far molecularly identified in brain are the brain Na⁺ channels (BNaCs). RT-PCRanalyses demonstrated the presence of mRNA for either BNaC1 or BNaC2 inthese tumors and in normal astrocytes. These results indicate that thefunctional expression of amiloride-sensitive Na⁺ currents is a characteristicfeature of malignant brain tumor cells and that this pathway may be apotentially useful target for therapeutic intervention.

     

High expression of integrin αvβ3 enables uptake of targeted fluorescent probes into ovarian cancer cells and tumors

The cell line OVCAR-4 was recently ranked as one of the most representative cell lines for high grade serous ovarian cancer (HGSOC). However, little work has been done to assess the susceptibility of OVCAR-4 cells and tumors to the more common types of molecular targeting. Proteome profiles suggest OVCAR-4 express high levels of integrin αvβ3 receptors. Using flow cytometry with fluorescent antibodies we determined that OVCAR-4 cells have high number of integrin αvβ3 receptors ([9.8?±?2.5]?×?10⁴/cell) compared to the well-characterized cell line U87-MG ([5.2?±?1.4]?×?10⁴/cell). However, OVCAR-4 cells also have roughly three times the surface area of U87-MG cells, so the average αvβ3 receptor density is actually lower (11?±?3 versus 18?±?6?receptors/µm²). A series of new fluorescent molecular probes was prepared with structures comprised of a deep-red squaraine fluorophore (～680?nm emission) covalently attached to zero, one, or two cyclic pentapeptide cRGD sequences for integrin targeting. Microscopy studies showed that uptake of the divalent probe into cultured OVCAR-4 cells was 2.2?±?0.4 higher than the monovalent probe, which in turn was 2.2?±?0.4 higher than the untargeted probe. This probe targeting trend was also seen in OVCAR-4 mouse tumor models. The results suggest that clinically relevant OVCAR-4 cells can be targeted using molecular probes based on αvβ3 integrin receptor antagonists such as the cRGD peptide. Furthermore, deep-red fluorescent cRGD-squaraine probes have potential as targeted stains of cancerous tissue associated with HGSOC in surgery and pathology settings.