IL-10-induced modulation of macrophage polarization suppresses outer-blood-retinal barrier disruption in the streptozotocin-induced early diabetic retinopathy mouse model.

Diabetic retinopathy (DR) is associated with ocular inflammation leading to retinal barrier breakdown, vascular leakage, macular edema, and vision loss. DR is not only a microvascular disease but also involves retinal neurodegeneration, demonstrating that pathological changes associated with neuroinflammation precede microvascular injury in early DR. Macrophage activation plays a central role in neuroinflammation. During DR, the inflammatory response depends on the polarization of retinal macrophages, triggering pro-inflammatory (M1) or anti-inflammatory (M2) activity. This study aimed to determine the role of macrophages in vascular leakage through the tight junction complexes of retinal pigment epithelium, which is the outer blood-retinal barrier (BRB). Furthermore, we aimed to assess whether interleukin-10 (IL-10), a representative M2-inducer, can decrease inflammatory macrophages and alleviate outer-BRB disruption. We found that modulation of macrophage polarization affects the structural and functional integrity of ARPE-19 cells in a co-culture system under high-glucose conditions. Furthermore, we demonstrated that intravitreal IL-10 injection induces an increase in the ratio of anti-inflammatory macrophages and effectively suppresses outer-BRB disruption and vascular leakage in a mouse model of early-stage streptozotocin-induced diabetes. Our results suggest that modulation of macrophage polarization by IL-10 administration during early-stage DR has a promising protective effect against outer-BRB disruption and vascular leakage. This finding provides valuable insights for early intervention in DR.

Mitochondrial transplantation attenuates oligomeric amyloid-beta-induced mitochondrial dysfunction and tight junction protein destruction in retinal pigment epithelium.

Transplantation of mitochondria derived from mesenchymal stem cells (MSCs) has emerged as a new treatment method to improve mitochondrial dysfunction and alleviate cell impairment. Interest in using extrinsic mitochondrial transplantation as a therapeutic approach has been increasing because it has been confirmed to be effective in treating various diseases related to mitochondrial dysfunction, including ischemia, cardiovascular disease, and toxic damage. To support this application, we conducted an experiment to deliver external mitochondria to retinal pigment epithelial cells treated with oligomeric amyloid-beta (oAß). Externally delivered amyloid-beta internalizes into cells and interacts with mitochondria, resulting in mitochondrial dysfunction and intracellular damage, including increased reactive oxygen species and destruction of tight junction proteins. Externally delivered mitochondria were confirmed to alleviate mitochondrial dysfunction and tight junction protein disruption as well as improve internalized oAß clearance. These results were also confirmed in a mouse model in vivo. Overall, these findings indicate that the transfer of external mitochondria isolated from MSCs has potential as a new treatment method for age-related macular degeneration, which involves oAß-induced changes to the retinal pigment epithelium.

Activation of Lysosomal Function Ameliorates Amyloid-ß-Induced Tight Junction Disruption in the Retinal Pigment Epithelium.

Accumulation of pathogenic amyloid-ß disrupts the tight junction of retinal pigment epithelium (RPE), one of its senescence-like structural alterations. In the clearance of amyloid-ß, the autophagy-lysosome pathway plays the crucial role. In this context, mammalian target of rapamycin (mTOR) inhibits the process of autophagy and lysosomal degradation, acting as a potential therapeutic target for age-associated disorders. However, efficacy of targeting mTOR to treat age-related macular degeneration remains largely elusive. Here, we validated the therapeutic efficacy of the mTOR inhibitors, Torin and PP242, in clearing amyloid-ß by inducing the autophagy-lysosome pathway in a mouse model with pathogenic amyloid-ß with tight junction disruption of RPE, which is evident in dry age-related macular degeneration. High concentration of amyloid-ß oligomers induced autophagy-lysosome pathway impairment accompanied by the accumulation of p62 and decreased lysosomal activity in RPE cells. However, Torin and PP242 treatment restored the lysosomal activity via activation of LAMP2 and facilitated the clearance of amyloid-ß in vitro and in vivo. Furthermore, clearance of amyloid-ß by Torin and PP242 ameliorated the tight junction disruption of RPE in vivo. Overall, our findings suggest mTOR inhibition as a new therapeutic strategy for the restoration of tight junctions in age-related macular degeneration.

Isolation and Characterization of the Primary Marmoset (Callithrix jacchus) Retinal Pigment Epithelial Cells.

Marmosets have emerged as a valuable primate model in ophthalmic research due to their similarity to the human visual system and their potential for generating transgenic models to advance the development of therapies. In this study, we isolated and cultured primary retinal pigment epithelium (RPE) cells from marmosets to investigate the mechanisms underlying RPE dysfunction in aging and age-related macular degeneration (AMD). We confirmed that our culture conditions and materials supported the formation of RPE monolayers with functional tight junctions that closely resembled the in vivo RPE. Since serum has been shown to induce epithelial-mesenchymal transition (EMT) in RPE cells, we compared the effects of fetal bovine serum (FBS) with serum-free supplements B27 on transepithelial electrical resistance (TER), cell proliferation, and morphological characteristics. Additionally, we assessed the age-related morphological changes of in vivo and primary RPE cells. Our results indicate that primary marmoset RPE cells exhibit in vivo-like characteristics, while cells obtained from an older donor show evidence of aging, including a failure to form a polarized monolayer, low TER, and delayed cell cycle. In conclusion, our primary marmoset RPE cells provide a reliable in vitro model for developing novel therapeutics for visual-threatening disorders such as AMD, which can be used before animal experiments using marmosets.

Inhibition of protein arginine deiminase II suppresses retinoblastoma in orthotopic transplantation in mice.

Chemotherapies are used for treating retinoblastoma; however, numerous patients suffer from recurrence or symptoms due to chemotherapy, which emphasizes the need for alternative therapeutic strategies. The present study demonstrated that protein arginine deiminase Ⅱ (PADI2) was highly expressed in human and mouse retinoblastoma tissues due to the overexpression of E2 factor (E2F). By inhibiting PADI2 activity, the expression of phosphorylated AKT was reduced, and cleaved poly (ADP­ribose) polymerase level was increased, leading to induced apoptosis. Similar results were obtained in orthotopic mouse models with reduced tumor volumes. In addition, BB­Cl­amidine showed low toxicity in vivo. These results suggested that PADI2 inhibition has potential clinical translation. Furthermore, the present study highlights the potential of epigenetic approaches to target RB1­deficient mutations at the molecular level. The current findings provide new insights into the importance of retinoblastoma intervention by managing PADI2 activity according to the treatment of specific inhibitors and depletion approaches in vitro and in orthotopic mouse models.

Visual function restoration in a mouse model of Leber congenital amaurosis via therapeutic base editing.

Leber congenital amaurosis (LCA), an inherited retinal degeneration, causes severe visual dysfunction in children and adolescents. In patients with LCA, pathogenic variants, such as RPE65, are evident in specific genes, related to the functions of retinal pigment epithelium and photoreceptors. In contrast to the original Cas9, base editing tools can correct pathogenic substitutions without generation of DNA double-stranded breaks (DSBs). In this study, dual adeno-associated virus (AAV) vectors containing split adenine base editors (ABEs) with trans-splicing intein were prepared for in vivo base editing in retinal degeneration of 12 (rd12) mice, an animal model of LCA, possessing a nonsense mutation of C to T transition in the Rpe65 gene (p.R44X). Subretinal injection of AAV-ABE in retinal pigment epithelial cells of rd12 mice resulted in an A to G transition. The on-target editing was sufficient for recovery of wild-type mRNA, RPE65 protein, and light-induced electrical responses from the retina. Compared with our previous therapeutic editing strategies using Cas9 and prime editing, or with the gene transfer strategy shown in the current study, our results suggest that, considering the editing efficacy and functional recovery, ABEs could be a strong, reliable method for correction of pathogenic variants in the treatment of LCA.

Application of Base Editor-Mediated Genome Editing in Mouse Retina.

Base editor is a newly developed genome editing technology that enables conversion of single nucleotides without DNA double-strand breaks (DSB) and maintains a low rate of insertion-deletion (INDEL) errors. With these flexibility and safety, base editor has been widely used in many fields, including inherited retinal disease. The majority of retinal genome editing requires intravitreal and subretinal injection delivery of the therapeutic vector in order to transduce the target cells. Here, we provide an application guide of base editor as performed in the mouse retina.

Enhancement of Gene Editing and Base Editing with Therapeutic Ribonucleoproteins through In Vivo Delivery Based on Absorptive Silica Nanoconstruct.

Key to the widespread and secure application of genome editing tools is the safe and effective delivery of multiple components of ribonucleoproteins (RNPs) into single cells, which remains a biological barrier to their clinical application. To overcome this issue, a robust RNP delivery platform based on a biocompatible sponge-like silica nanoconstruct (SN) for storing and directly delivering therapeutic RNPs, including Cas9 nuclease RNP (Cas9-RNP) and base editor RNP (BE-RNP) is designed. Compared with commercialized material such as lipid-based methods, up to 50-fold gene deletion and 10-fold base substitution efficiency is obtained with a low off-target efficiency by targeting various cells and genes. In particular, gene correction is successfully induced by SN-based delivery through intravenous injection in an in vivo solid-tumor model and through subretinal injection in mouse eye. Moreover, because of its low toxicity and high biodegradability, SN has negligible effect on cellular function of organs. As the engineered SN can overcome practical challenges associated with therapeutic RNP application, it is strongly expected this platform to be a modular RNPs delivery system, facilitating in vivo gene deletion and editing.

Establishment and Characterization of Carboplatin-Resistant Retinoblastoma Cell Lines.

Carboplatin-based chemotherapy is the primary treatment option for the management of retinoblastoma, an intraocular malignant tumor observed in children. The aim of the present study was to establish carboplatin-resistant retinoblastoma cell lines to facilitate future research into the treatment of chemoresistant retinoblastoma. In total, two retinoblastoma cell lines, Y79 and SNUOT-Rb1, were treated with increasing concentrations of carboplatin to develop the carboplatin-resistant retinoblastoma cell lines (termed Y79/CBP and SNUOT-Rb1/CBP, respectively). To verify resistance to carboplatin, the degree of DNA fragmentation and the expression level of cleaved caspase-3 were evaluated in the cells, following carboplatin treatment. In addition, the newly developed carboplatin-resistant retinoblastoma cells formed in vivo intraocular tumors more effectively than their parental cells, even after the intravitreal injection of carboplatin. Interestingly, the proportion of cells in the G0/G1 phase was higher in Y79/CBP and SNUOT-Rb1/CBP cells than in their respective parental cells. In line with these data, the expression levels of cyclin D1 and cyclin D3 were decreased, whereas p18 and p27 expression was increased in the carboplatin-resistant cells. In addition, the expression levels of genes associated with multidrug resistance were increased. Thus, these carboplatin-resistant cell lines may serve as a useful tool in the study of chemoresistance in retinoblastoma and for the development potential therapeutics.

Self-Plugging Microneedle (SPM) for Intravitreal Drug Delivery.

Intravitreal injection (IVI) is a common technology which is used to treat ophthalmic diseases inside eyeballs by delivering various drugs into the vitreous cavity using hypodermic needles. However, in some cases, there are possible side effects such as ocular tissue damage due to repeated injection or eyeball infection through the hole created during the needle retraction process. The best scenario of IVI is a one-time injection of drugs without needle retraction, keeping the system of the eyeball closed. Microneedles (MNs) have been applied to ocular tissues over 10 years, and no serious side effects on ocular tissue due to MN injection have been reported. Therefore, a self-plugging MN (SPM) is developed to perform intraocular drug delivery and to seal the scleral puncture simultaneously. The SPMs are fabricated by a thermal drawing process and then coated with a polymeric carrier of drugs and a hydrogel-based scleral plugging component. Each coated functional layer is characterized and demonstrated by in vitro and ex vivo experiments. Finally, in vivo tests using a porcine model confirms prompt sealing of SPM and sustained intraocular drug delivery.

Application of prime editing to the correction of mutations and phenotypes in adult mice with liver and eye diseases.

The use of prime editing-a gene-editing technique that induces small genetic changes without the need for donor DNA and without causing double strand breaks-to correct pathogenic mutations and phenotypes needs to be tested in animal models of human genetic diseases. Here we report the use of prime editors 2 and 3, delivered by hydrodynamic injection, in mice with the genetic liver disease hereditary tyrosinemia, and of prime editor 2, delivered by an adeno-associated virus vector, in mice with the genetic eye disease Leber congenital amaurosis. For each pathogenic mutation, we identified an optimal prime-editing guide RNA by using cells transduced with lentiviral libraries of guide-RNA-encoding sequences paired with the corresponding target sequences. The prime editors precisely corrected the disease-causing mutations and led to the amelioration of the disease phenotypes in the mice, without detectable off-target edits. Prime editing should be tested further in more animal models of genetic diseases.

Giant Y79 retinoblastoma cells contain functionally active T-type calcium channels.

Retinoblastoma is the most common malignant intraocular tumor in children. Y79 human retinoblastoma cells are in vitro models of retinal tumors used for drug screening. Undifferentiated Y79 cells originate from a primitive multi-potential neuroectodermal cell and express neuronal and glial properties. However, the nature of cellular heterogeneity in Y79 cells is unclear because functional methods to characterize neurons or glial cells have not been employed to Y79 cells. Here, we perform patch-clamp recordings to characterize electrophysiological properties in retinoblastoma cells. We identified a population of large-sized Y79 cells (i.e., giant cells, ~ 40-µm diameter), hyperpolarized resting membrane potential (-54 mV), and low input resistance (~ 600 MΩ), indicating electrically mature cells. We also found that giant Y79 cells contain increased density of T-type calcium channels. Finally, we found that T-type calcium channels are active only in giant cells suggesting that cancer treatments aimed to prevent calcium influx in retinoblastomas should be tested in giant cells.

High-purity production and precise editing of DNA base editing ribonucleoproteins.

Ribonucleoprotein (RNP) complex-mediated base editing is expected to be greatly beneficial because of its reduced off-target effects compared to plasmid- or viral vector-mediated gene editing, especially in therapeutic applications. However, production of recombinant cytosine base editors (CBEs) or adenine base editors (ABEs) with ample yield and high purity in bacterial systems is challenging. Here, we obtained highly purified CBE/ABE proteins from a human cell expression system and showed that CBE/ABE RNPs exhibited different editing patterns (i.e., less conversion ratio of multiple bases to single base) compared to plasmid-encoded CBE/ABE, mainly because of the limited life span of RNPs in cells. Furthermore, we found that off-target effects in both DNA and RNA were greatly reduced for ABE RNPs compared to plasmid-encoded ABE. We ultimately applied NG PAM-targetable ABE RNPs to in vivo gene correction in retinal degeneration 12 (rd12) model mice.

Corneal lymphangiogenesis in dry eye disease is regulated by substance P/neurokinin-1 receptor system through controlling expression of vascular endothelial growth factor receptor 3.

PURPOSE: To evaluate the role of substance P (SP)/neurokinin-1 receptor (NK1R) system in the regulation of pathologic corneal lymphangiogenesis in dry eye disease (DED). METHODS: Immunocytochemistry, angiogenesis assay, and Western blot analysis of human dermal lymphatic endothelial cells (HDLECs) were conducted to assess the involvement of SP/NK1R system in lymphangiogenesis. DED was induced in wild-type C57BL/6 J mice using controlled-environment chamber without scopolamine. Immunohistochemistry, corneal fluorescein staining, and phenol red thread test were used to evaluate the effect of SP signaling blockade in the corneal lymphangiogenesis. The expression of lymphangiogenic factors in the corneal and conjunctival tissues of DED mouse model was quantified by real-time polymerase chain reaction. RESULTS: NK1R expression and pro-lymphangiogenic property of SP/NK1R system in HDLECs were confirmed by Western blot analysis and angiogenesis assay. Blockade of SP signaling with L733,060, an antagonist of NK1R, or NK1R-targeted siRNA significantly inhibited lymphangiogenesis and expression of vascular endothelial growth factor (VEGF) receptor 3 stimulated by SP in HDLECs. NK1R antagonist also suppressed pathological corneal lymphangiogenesis and ameliorated the clinical signs of dry eye in vivo. Furthermore, NK1R antagonist effectively suppressed the lymphangiogenic factors, including VEGF-C, VEGF-D, and VEGF receptor 3 in the corneal and conjunctival tissues of DED. CONCLUSIONS: SP/NK1R system promotes lymphangiogenesis in vitro and NK1R antagonism suppresses pathologic corneal lymphangiogenesis in DED in vivo.

Antitumor Activity of Novel Signal Transducer and Activator of Transcription 3 Inhibitors on Retinoblastoma.

Signal transducer and activator of transcription 3 (STAT3) is a plausible therapeutic target in the treatment of retinoblastoma, the most common intraocular malignant tumor in children. STAT3, a transcription factor of several genes related to tumorigenesis, is activated in retinoblastoma tumors as well as other cancers. In this study, we investigated the structure-activity relationship of a library of STAT3 inhibitors, including a novel series of derivatives of the previously reported compound with a Michael acceptor (compound 1). We chose two novel STAT3 inhibitors, compounds 11 and 15, from the library based on their inhibitory effects on the phosphorylation and transcription activity of STAT3. These STAT3 inhibitors effectively suppressed the phosphorylation of STAT3 and inhibited the expression of STAT3-related genes CCND1, CDKN1A, BCL2, BCL2L1, BIRC5, MYC, MMP1, MMP9, and VEGFA Intraocularly administered STAT3 inhibitors decreased the degree of tumor formation in the vitreous cavity of BALB/c nude mice of an orthotopic transplantation model. It is noteworthy that compounds 11 and 15 did not induce in vitro and in vivo toxicity on retinal constituent cells and retinal tissues, respectively, despite their potent antitumor effects. We suggest that these novel STAT3 inhibitors be used in the treatment of retinoblastoma. SIGNIFICANCE STATEMENT: The current study suggests the novel STAT3 inhibitors with Michael acceptors possess antitumor activity on retinoblastoma, the most common intraocular cancer in children. Based on detailed structure-activity relationship studies, we found a 4-fluoro and 3-trifluoro analog (compound 11) and a monochloro analog (compound 15) of the parental compound (compound 1) inhibited STAT3 phosphorylation, leading to suppressed retinoblastoma in vitro and in vivo.

Specific ablation of PDGFRß-overexpressing pericytes with antibody-drug conjugate potently inhibits pathologic ocular neovascularization in mouse models.

Background: Crosstalk between pericytes and endothelial cells is critical for ocular neovascularization. Endothelial cells secrete platelet-derived growth factor (PDGF)-BB and recruit PDGF receptor ß (PDGFRß)-overexpressing pericytes, which in turn cover and stabilize neovessels, independent of vascular endothelial growth factor (VEGF). Therapeutic agents inhibiting PDGF-BB/PDGFRß signaling were tested in clinical trials but failed to provide additional benefits over anti-VEGF agents. We tested whether an antibody-drug conjugate (ADC) - an engineered monoclonal antibody linked to a cytotoxic agent - could selectively ablate pericytes and suppress retinal and choroidal neovascularization. Methods: Immunoblotting, flow cytometry, cell viability test, and confocal microscopy were conducted to assess the internalization and cytotoxic effect of ADC targeting mPDGFRß in an in vitro setting. Immunofluorescence staining of whole-mount retinas and retinal pigment epithelium-choroid-scleral complexes, electroretinography, and OptoMotry test were used to evaluate the effect and safety of ADC targeting mPDGFRß in the mouse models of pathologic ocular neovascularization. Results: ADC targeting mPDGFRß is effectively internalized into mouse brain vascular pericytes and showed significant cytotoxicity compared with the control ADC. We also show that specific ablation of PDGFRß-overexpressing pericytes using an ADC potently inhibits pathologic ocular neovascularization in mouse models of oxygen-induced retinopathy and laser-induced choroidal neovascularization, while not provoking generalized retinal toxicity. Conclusion: Our results suggest that removing PDGFRß-expressing pericytes by an ADC targeting PDGFRß could be a potential therapeutic strategy for pathologic ocular neovascularization.

Histamine causes an imbalance between pro-angiogenic and anti-angiogenic factors in the retinal pigment epithelium of diabetic retina via H4 receptor/p38 MAPK axis.

INTRODUCTION: Systemic histaminergic activity is elevated in patients with diabetes mellitus. There are a few studies suggesting that histamine is implicated in the pathogenesis of diabetes, but the exact role of histamine in the development of diabetic retinopathy is unclear. The aim of this study was to investigate the role of histamine receptor H4 (HRH4) in the regulation of retinal pigment epithelium (RPE)-derived pro-angiogenic and anti-angiogenic factors under diabetic conditions. RESEARCH DESIGN AND METHODS: The levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), histamine and histidine decarboxylase (HDC) in the serum and vitreous samples of patients with diabetes were compared with those of patients without diabetes. The effect of hyperglycemia on expression levels of HRH4, VEGF, IL-6 and pigment epithelium-derived factor (PEDF) in the RPE was determined. The role of HRH4 in high glucose-induced regulation of VEGF, IL-6 and PEDF in ARPE-19 cells and the underlying regulatory mechanism were verified using an RNA interference-mediated knockdown study. RESULTS: The serum and vitreous levels of VEGF, IL-6, histamine and HDC were more increased in patients with diabetic retinopathy than in patients without diabetes. HRH4 was overexpressed in RPE both in vitro and in vivo. Histamine treatment upregulated VEGF and IL-6 and downregulated PEDF expression in ARPE-19 cells cultivated under hyperglycemic conditions. Hyperglycemia-induced phosphorylation of p38 and subsequent upregulation of VEGF and IL-6 and downregulation of PEDF were dampened by small interfering RNA-mediated knockdown of HRH4 in ARPE-19 cells. CONCLUSIONS: Taken together, HRH4 was a critical regulator of VEGF, IL-6 and PEDF in the RPE under hyperglycemic conditions and the p38 mitogen-activated protein kinase pathway mediated this regulatory mechanism.

Intracellular amyloid-ß disrupts tight junctions of the retinal pigment epithelium via NF-κB activation.

Drusen are focal deposits between the retinal pigment epithelium (RPE) and Bruch's membrane in the retina of patients with age-related macular degeneration. Amyloid-ß is one of the important components of drusen, which leads to local inflammation. Furthermore, intracellular amyloid-ß disrupts tight junctions of the RPE. However, the intracellular mechanisms linking intracellular amyloid-ß and tight-junction disruption are not clear. In this study, intracellular amyloid-ß oligomers activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65, leading to the disorganization of tight junctions of the RPE in mice after subretinal injection of amyloid-ß. Amyloid-ß also triggered NF-κB activation in the RPE cells in confluent culture, which was inhibited by the suppression of the advanced glycosylation end product-specific receptor. NF-κB inhibition by an IκB kinase inhibitor prevented the suppression of expression of tight-junction proteins, zonula occuludens-1 and occludin in RPE cells. In addition, tight-junction complexes remained intact in the RPE of mice with NF-κB inhibition, although there were intracellular amyloid-ß oligomers. These data suggested that NF-κB inhibition might be a therapeutic approach to prevent amyloid-ß-mediated tight-junction disruption.

CRISPR-Cas9-mediated therapeutic editing of Rpe65 ameliorates the disease phenotypes in a mouse model of Leber congenital amaurosis.

Leber congenital amaurosis (LCA), one of the leading causes of childhood-onset blindness, is caused by autosomal recessive mutations in several genes including RPE65. In this study, we performed CRISPR-Cas9-mediated therapeutic correction of a disease-associated nonsense mutation in Rpe65 in rd12 mice, a model of human LCA. Subretinal injection of adeno-associated virus carrying CRISPR-Cas9 and donor DNA resulted in >1% homology-directed repair and ~1.6% deletion of the pathogenic stop codon in Rpe65 in retinal pigment epithelial tissues of rd12 mice. The a- and b-waves of electroretinograms were recovered to levels up to 21.2 ± 4.1% and 39.8 ± 3.2% of their wild-type mice counterparts upon bright stimuli after dark adaptation 7 months after injection. There was no definite evidence of histologic perturbation or tumorigenesis during 7 months of observation. Collectively, we present the first therapeutic correction of an Rpe65 nonsense mutation using CRISPR-Cas9, providing new insight for developing therapeutics for LCA.

Development of a patient-derived xenograft model of glioblastoma via intravitreal injection in mice.

Currently, the two primary patient-derived xenograft (PDX) models of glioblastoma are established through intracranial or subcutaneous injection. In this study, a novel PDX model of glioblastoma was developed via intravitreal injection to facilitate tumor formation in a brain-mimicking microenvironment with improved visibility and fast development. Glioblastoma cells were prepared from the primary and recurrent tumor tissues of a 39-year-old female patient. To demonstrate the feasibility of intracranial tumor formation, U-87 MG and patient-derived glioblastoma cells were injected into the brain parenchyma of Balb/c nude mice. Unlike the U-87 MG cells, the patient-derived glioblastoma cells failed to form intracranial tumors until 6 weeks after tumor cell injection. In contrast, the patient-derived cells effectively formed intraocular tumors, progressing from plaques at 2 weeks to masses at 4 weeks after intravitreal injection. The in vivo tumors exhibited the same immunopositivity for human mitochondria, GFAP, vimentin, and nestin as the original tumors in the patient. Furthermore, cells isolated from the in vivo tumors also demonstrated morphology similar to that of their parental cells and immunopositivity for the same markers. Overall, a novel PDX model of glioblastoma was established via the intravitreal injection of tumor cells. This model will be an essential tool to investigate and develop novel therapeutic alternatives for the treatment of glioblastoma.