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Epidermal growth factor receptor (EGFR)-targeted monoclonal antibodies, including cetuximab and panitumumab, are used to treat metastatic colorectal cancer (mCRC). However, this treatment is only effective for a small subset of mCRC patients positive for the wild-type KRAS GTPase. GC1118 is a novel, fully humanized anti-EGFR IgG1 antibody that displays potent inhibitory effects on high-affinity EGFR ligand-induced signaling and enhanced antibody-mediated cytotoxicity. In this study, using 51 CRC patient-derived xenografts (PDXs), we showed that KRAS mutants expressed remarkably elevated autocrine levels of high-affinity EGFR ligands compared with wild-type KRAS. In three KRAS-mutant CRCPDXs, GC1118 was more effective than cetuximab, whereas the two agents demonstrated comparable efficacy against three wild-type KRAS PDXs. Persistent phosphatidylinositol-3-kinase (PI3K)/AKT signaling was thought to underlie resistance to GC1118. In support of these findings, a preliminary improved anti-cancer response was observed in a CRC PDX harboring mutated KRAS with intrinsically high AKT activity using GC1118 combined with the dual PI3K/mammalian target of rapamycin (mTOR)/AKT inhibitor BEZ-235, without observed toxicity. Taken together, the superior antitumor efficacy of GC1118 alone or in combination with PI3K/mTOR/AKT inhibitors shows great therapeutic potential for the treatment of KRAS-mutant mCRC with elevated ratios of high- to low-affinity EGFR ligands and PI3K-AKT pathway activation.
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Anticorpos Monoclonais Humanizados/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Cetuximab/farmacologia , Cetuximab/uso terapêutico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Receptores ErbB/imunologia , Feminino , Humanos , Camundongos , Camundongos Nus , Mutação , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
The receptor tyrosine kinase c-Met plays critical roles in promoting tumor growth, invasion, metastasis, and angiogenesis in various types of cancer and is a promising therapeutic target. The development of a species cross-reactive therapeutic antibody could provide useful to comprehensive preclinical assessment in animal models. Towards this goal, we developed human/mouse cross-reactive c-Met antibodies using an antibody phage library. IRCR201, a c-Met antibody with species cross-reactivity, successfully inhibited the HGF/c-Met signaling pathway via degradation of c-Met and disruption of the binding with its partners, and demonstrated strong in vivo antitumor activity. In pharmacokinetic analysis, IRCR201 exhibited a nonlinear pharmacokinetic profile and showed rapid serum clearance at low dosage. Ex vivo fluorescence imaging and immunohistochemistry demonstrated strong tumor accumulation of IRCR201. Hepatotoxicity analysis revealed that IRCR201 does not significantly affect primary human and mouse hepatocytes. Serum chemistry analysis demonstrated that the alanine aminotransferase serum level was elevated in mice treated with 30 mg/kg IRCR201 than in PBS-treated mice, whereas the levels of aspartate aminotransferase and blood urea nitrogen did not significantly differ. Thus, IRCR201 is a potent therapeutic antibody that can disrupt the HGF/c-Met signaling axis and its species cross-reactivity would enable to evaluate precise biological activity in animal models.
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Anticorpos Antineoplásicos/farmacologia , Anticorpos Neutralizantes/farmacologia , Antineoplásicos/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Neoplasias Gástricas/tratamento farmacológico , Animais , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Reações Cruzadas , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Feminino , Expressão Gênica , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/imunologia , Humanos , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neuroglia/imunologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/imunologia , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hepatocyte growth factor receptor (HGFR, c-Met) is an essential member of the receptor tyrosine kinase (RTK) family that is often dysregulated during tumor progression, driving a malignant phenotypic state and modulating important cellular functions including tumor growth, invasion, metastasis, and angiogenesis, providing a strong rationale for targeting HGF/c-Met signaling axis in cancer therapy. Based on its protumorigenic potentials, we developed IRCR201, a potent antagonistic antibody targeting the plexin-semaphorin-integrin (PSI) domain of c-Met, using synthetic human antibody phage libraries. We characterized and evaluated the biochemical properties and tumor inhibitory effect of IRCR201 in vitro and in vivo. IRCR201 is a novel fully-human bivalent therapeutic antibody that exhibits cross-reactivity against both human and mouse c-Met proteins with high affinity and specificity. IRCR201 displayed low agonist activity and rapidly depleted total c-Met protein via the lysosomal degradation pathway, inhibiting c-Met-dependent downstream activation and attenuating cellular proliferation in various c-Met-expressing cancer cells. In vivo tumor xenograft models also demonstrated the superior tumor inhibitory responsiveness of IRCR201. Taken together, IRCR201 provides a promising therapeutic agent for c-Met-positive cancer patients through suppressing the c-Met signaling pathway and tumor growth.
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Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Antineoplásicos/farmacologia , Proteínas Proto-Oncogênicas c-met/imunologia , Células A549 , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/imunologia , Anticorpos Neutralizantes/imunologia , Antineoplásicos/imunologia , Apoptose/efeitos dos fármacos , Moléculas de Adesão Celular/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reações Cruzadas , Mapeamento de Epitopos , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/imunologia , Humanos , Imuno-Histoquímica , Integrinas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Tecido Nervoso/imunologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Semaforinas/imunologia , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: KNP-301 is a bi-specific fragment crystallizable region (Fc) fusion protein, which inhibits both C3b and vascular endothelial growth factor (VEGF) simultaneously for patients with late-stage age-related macular degeneration (AMD). The present study evaluated in vitro potency, in vivo efficacy, intravitreal pharmacokinetics (IVT PK), and injectability of KNP-301. METHODS: C3b and VEGF binding of KNP-301 were assessed by surface plasmon resonance (SPR) and enzyme-linked immunosorbent assay (ELISA), and cellular bioassays. A laser-induced choroidal neovascularization (CNV) model and a sodium iodate-induced nonexudative AMD model were used to test the in vivo efficacy of mouse surrogate of KNP-301. Utilizing fluorescein angiography (FA) and spectral-domain optical coherence tomography (SD-OCT) scans, the reduction in disease lesions were analyzed in a CNV mouse model. In the nonexudative AMD mouse model, outer nuclear layer (ONL) was assessed by immunofluorescence staining. Lastly, intravitreal pharmacokinetic study was conducted with New Zealand white rabbits via IVT administration of KNP-301 and injectability of KNP-301 was examined by a viscosity test at high concentrations. RESULTS: KNP-301 bound C3b selectively, which resulted in a blockade of the alternative pathway, not the classical pathway. KNP-301 also acted as a VEGF trap, impeding VEGF-mediate signaling. Our dual-blockade strategy was effective in both neovascular and nonexudative AMD models. Moreover, KNP-301 had an advantage of potentially less frequent dosing due to the long half-life in the intravitreal chamber. Our viscosity assessment confirmed that KNP-301 meets the criteria of the IVT injection. CONCLUSIONS: Unlike current therapies, KNP-301 is expected to cover patients with late-stage AMD of both neovascular and nonexudative AMD, and its long-term PK profile at the intravitreal chamber would allow convenience in the dosing interval of patients.
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Immunotherapy has emerged as a promising approach to cancer treatment that utilizes the potential of the immune system to precisely identify and eradicate cancerous cells. Despite significant progress in immunotherapy, innovative approaches are required to enhance the effectiveness and safety of these treatments. Interleukin-12 (IL-12), widely recognized for its essential function in immune responses, has been explored as a potential candidate for treating cancer. However, early attempts involving the systemic administration of IL-12 were ineffective, with significant adverse effects, thus underscoring the need for innovation. To address these challenges, we developed a therapeutic molecule that utilizes a single-chain IL-12 mutant (IL-12mut) linked to a tumor-targeting arm. Here, we describe the development of a highly effective IL-12-based TMEkine™ platform by employing a B-cell lymphoma model (termed CD20-IL-12mut). CD20-IL-12mut combined the attenuated activities of IL-12 with targeted delivery to the tumor, thereby maximizing therapeutic potential while minimizing off-target effects. Our results revealed that CD20-IL-12mut exhibited potent anticancer activity by inducing complete regression and generating immunological memory for tumor antigens. Collectively, our data provide a basis for additional research on CD20-IL-12mut as a potential treatment choice for patients with B-cell lymphomas such as non-Hodgkin's lymphoma.
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Imunoterapia , Interleucina-12 , Linfoma de Células B , Interleucina-12/genética , Interleucina-12/imunologia , Animais , Linfoma de Células B/terapia , Linfoma de Células B/imunologia , Imunoterapia/métodos , Humanos , Linhagem Celular Tumoral , Antígenos CD20/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Feminino , Memória ImunológicaRESUMO
While cancer immunotherapy has yielded encouraging outcomes in hematological malignancies, it has faced challenges in achieving the same level of effectiveness in numerous solid tumors, primarily because of the presence of immune-suppressive tumor microenvironments (TMEs). The immunosuppressive qualities of the TME have generated considerable interest, making it a focal point for treatments aimed at enhancing immune responses and inhibiting tumor progression. Fibroblast activation protein (FAP), an attractive candidate for targeted immunotherapy, is prominently expressed in the TME of various solid tumors. Interleukin-12 (IL-12), recognized as a key mediator of immune responses, has been explored as a potential candidate for cancer treatment. Nevertheless, initial efforts to administer IL-12 systemically demonstrated limited efficacy and notable side effects, emphasizing the necessity for innovation. To address these concerns, our molecules incorporated specific IL-12 mutations, called IL-12mut, which reduced toxicity. This study explored the therapeutic potential of the FAP-IL-12mut TMEkine™-a novel immunotherapeutic agent selectively engineered to target FAP-expressing cells in preclinical cancer models. Our preclinical results, conducted across diverse murine cancer models, demonstrated that FAP-IL-12mut significantly inhibits tumor growth, enhances immune cell infiltration, and promotes a shift toward a cytotoxic immune activation profile. These findings suggest that FAP-IL-12mut could offer effective cancer treatment strategies.
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Nigrospora (Xylariales, Apiosporaceae) consists of species of terrestrial plant endophytes and pathogens. Nigrospora has also been reported in marine environments such as mangroves, sea fans, and macroalgae. However, limited research has been conducted on Nigrospora associated with macroalgae. Here, we isolated Nigrospora species from three types of algae (brown, green, and red algae) from Korean islands (Chuja, Jeju, and Ulleung) based on phylogenetic analyses of multigenetic markers: the internal transcribed spacers (ITS), beta-tubulin (BenA), and translation elongation factor 1 (TEF1-α). A total of 17 Nigrospora strains were isolated from macroalgae and identified as nine distinct species. The majority of Nigrospora species (seven) were found on brown algae, followed by red algae (three), and then green algae (two). To our understanding, this study represents the first account of N. cooperae, N. covidalis, N. guilinensis, N. lacticolonia, N. osmanthi, N. pyriformis, and N. rubi occurring in marine environments. Additionally, this study provides the first report of the occurrence of N. cooperae, N. covidalis, N. guilinensis, N. lacticolonia, and N. osmanthi in South Korea. This study will provide valuable insights for future research exploring the functions of fungi in macroalgal communities.
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Intuitive and perceptual neuroprosthetic systems require a high degree of neural control and a variety of sensory feedback, but reliable neural interfaces for long-term use that maintain their functionality are limited. Here, a novel hybrid bionic interface is presented, fabricated by integrating a biological interface (regenerative peripheral nerve interface (RPNI)) and a peripheral neural interface to enhance the neural interface performance between a nerve and bionic limbs. This interface utilizes a shape memory polymer buckle that can be easily implanted on a severed nerve and make contact with both the nerve and the muscle graft after RPNI formation. It is demonstrated that this interface can simultaneously record different signal information via the RPNI and the nerve, as well as stimulate them separately, inducing different responses. Furthermore, it is shown that this interface can record naturally evoked signals from a walking rabbit and use them to control a robotic leg. The long-term functionality and biocompatibility of this interface in rabbits are evaluated for up to 29 weeks, confirming its promising potential for enhancing prosthetic control.
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Biônica , Nervos Periféricos , Animais , Coelhos , Eletromiografia , Nervos Periféricos/fisiologia , Próteses e Implantes , Regeneração Nervosa/fisiologiaRESUMO
BACKGROUND: Although temozolomide (TMZ) has been used as a standard adjuvant chemotherapeutic agent for primary glioblastoma (GBM), treating isocitrate dehydrogenase wild-type (IDH-wt) cases remains challenging due to intrinsic and acquired drug resistance. Therefore, elucidation of the molecular mechanisms of TMZ resistance is critical for its precision application. METHODS: We stratified 69 primary IDH-wt GBM patients into TMZ-resistant (n = 29) and sensitive (n = 40) groups, using TMZ screening of the corresponding patient-derived glioma stem-like cells (GSCs). Genomic and transcriptomic features were then examined to identify TMZ-associated molecular alterations. Subsequently, we developed a machine learning (ML) model to predict TMZ response from combined signatures. Moreover, TMZ response in multisector samples (52 tumor sectors from 18 cases) was evaluated to validate findings and investigate the impact of intra-tumoral heterogeneity on TMZ efficacy. RESULTS: In vitro TMZ sensitivity of patient-derived GSCs classified patients into groups with different survival outcomes (P = 1.12e-4 for progression-free survival (PFS) and 3.63e-4 for overall survival (OS)). Moreover, we found that elevated gene expression of EGR4, PAPPA, LRRC3, and ANXA3 was associated to intrinsic TMZ resistance. In addition, other features such as 5-aminolevulinic acid negative, mesenchymal/proneural expression subtypes, and hypermutation phenomena were prone to promote TMZ resistance. In contrast, concurrent copy-number-alteration in PTEN, EGFR, and CDKN2A/B was more frequent in TMZ-sensitive samples (Fisher's exact P = 0.0102), subsequently consolidated by multi-sector sequencing analyses. Integrating all features, we trained a ML tool to segregate TMZ-resistant and sensitive groups. Notably, our method segregated IDH-wt GBM patients from The Cancer Genome Atlas (TCGA) into two groups with divergent survival outcomes (P = 4.58e-4 for PFS and 3.66e-4 for OS). Furthermore, we showed a highly heterogeneous TMZ-response pattern within each GBM patient using in vitro TMZ screening and genomic characterization of multisector GSCs. Lastly, the prediction model that evaluates the TMZ efficacy for primary IDH-wt GBMs was developed into a webserver for public usage ( http://www.wang-lab-hkust.com:3838/TMZEP ). CONCLUSIONS: We identified molecular characteristics associated to TMZ sensitivity, and illustrate the potential clinical value of a ML model trained from pharmacogenomic profiling of patient-derived GSC against IDH-wt GBMs.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Farmacogenética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Glioma/genética , Resistencia a Medicamentos Antineoplásicos/genética , Fatores de Transcrição de Resposta de Crescimento PrecoceRESUMO
BACKGROUND: High-intensity focused ultrasound (HIFU) therapy has emerged as an option for skin rejuvenation. However, the application against photo-damaged skin remains obscure. This study evaluates the effect of HIFU against photoaged skin using a mouse model. METHODS: A total of 60 mice were used and divided into 3 groups; group 1: natural aging control group (n = 20), group 2: UVB irradiation group (n = 20), and group 3: UVB irradiation followed by HIFU treatment (n = 20). The evaluation was made grossly by analyzing wrinkles and histologically by performing H&E, Toluidine Blue, Masson's Trichrome, and immunohistochemistry for TGF-ß and MMP3. Imaging software was used to quantify the findings. RESULTS: Gross findings showed HIFU treated group 3 with similar findings with the control group supporting the rejuvenation effect for photo-aged skin. Histology findings with H&E show a significant reduction in skin thickness after HIFU treatment (60.115 units (group 2) vs. 40.853 units (group 3), p<0.05). Toluidine Blue and Masson's Trichrome showed improved collagen array and significantly increased distribution for group 3 over group 2 (272,879.88 units (group 2) vs. 533,805.78 units (group 3), p<0.05). Immunohistochemistry for TGF-ß showed a significantly higher value for group 3 (2.45450 units) over group 2 (0.58880 units) and MMP3 with a significantly lower value for group 3 (99,180 units) over group 2 (559,830 units) (p<0.05). CONCLUSIONS: The treatment of HIFU supports the rejuvenation effect for photoaged skin. Findings show that HIFU provides benefits of collagen formation and rearrangement by enhancing TGF-ß and inhibiting MMP3 activity. This study is the first animal study to show the direct effect of HIFU on photo-aged skin further supporting the use of HIFU in aging skin aiming to reverse the morphological effects of aging.
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Rejuvenescimento , Envelhecimento da Pele , Animais , Colágeno , Metaloproteinase 3 da Matriz , Cloreto de Tolônio , Fator de Crescimento Transformador betaRESUMO
PTEN is one of the most frequently altered tumor suppressor genes in malignant tumors. The dominant-negative effect of PTEN alteration suggests that the aberrant function of PTEN mutation might be more disastrous than deletion, the most frequent genomic event in glioblastoma (GBM). This study aimed to understand the functional properties of various PTEN missense mutations and to investigate their clinical relevance. The genomic landscape of PTEN alteration was analyzed using the Samsung Medical Center GBM cohort and validated via The Cancer Genome Atlas dataset. Several hotspot mutations were identified, and their subcellular distributions and phenotypes were evaluated. We established a library of cancer cell lines that overexpress these mutant proteins using the U87MG and patient-derived cell models lacking functional PTEN. PTEN mutations were categorized into two major subsets: missense mutations in the phosphatase domain and truncal mutations in the C2 domain. We determined the subcellular compartmentalization of four mutant proteins (H93Y, C124S, R130Q, and R173C) from the former group and found that they had distinct localizations; those associated with invasive phenotypes ('edge mutations') localized to the cell periphery, while the R173C mutant localized to the nucleus. Invasive phenotypes derived from edge substitutions were unaffected by an anti-PI3K/Akt agent but were disrupted by microtubule inhibitors. PTEN mutations exhibit distinct functional properties regarding their subcellular localization. Further, some missense mutations ('edge mutations') in the phosphatase domain caused enhanced invasiveness associated with dysfunctional cytoskeletal assembly, thus suggesting it to be a potent therapeutic target.
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Glioblastoma/genética , Oncogenes/genética , PTEN Fosfo-Hidrolase/metabolismo , Humanos , MutaçãoRESUMO
c-Met, as a receptor expressed on the cell membrane, contributes to the growth and metastasis of tumors, as well as angiogenesis, mainly through the hepatocyte growth factor (HGF)/c-Met axis during tumor progression. Although several c-Met inhibitors, including small molecules and monoclonal antibody inhibitors, are currently being investigated, their clinical outcomes have not been promising. Development of an antibody-drug conjugate (ADC) against c-Met could be an attractive therapeutic strategy that would provide superior antitumor efficacy with broad-spectrum c-Met expression levels. In the present study, site-specific drug-conjugate technology was applied to develop an ADC using the human-mouse cross-reactive c-Met antibody and a prodrug pyrrolobenzodiazepine (PBD). The toxin payload was uniformly conjugated to the light-chain C-terminus of the native cIRCR201 antibody (drug-to-antibody ratio = 2), as confirmed using LC-MS. Using a high-throughput screening system, we found that cIRCR201-dPBD exhibited varying sensitivities depending on the expression levels of c-Met, and it induced receptor-mediated endocytosis and toxin-mediated apoptosis in 47 different cancer cell lines. cIRCR201-dPBD also showed significant antitumor activity on the MET-amplified cancer cells using in vivo xenograft models. Therefore, cIRCR201-dPBD could be a promising therapeutic strategy for tumors with c-Met expression.
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We aimed to evaluate the preclinical efficacy of GC1118, a novel anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb), against glioblastoma (GBM) tumors using patient-derived xenograft (PDX) models. A total of 15 distinct GBM PDX models were used to evaluate the therapeutic efficacy of GC1118. Genomic data derived from PDX models were analyzed to identify potential biomarkers associated with the anti-tumor efficacy of GC1118. A patient-derived cell-based high-throughput drug screening assay was performed to further validate the efficacy of GC1118. Compared to cetuximab, GC1118 exerted comparable growth inhibitory effects on the GBM tumors in the PDX models. We confirmed that GC1118 accumulated within the tumor by crossing the blood-brain barrier in in vivo specimens and observed the survival benefit in GC1118-treated intracranial models. Genomic analysis revealed high EGFR amplification as a potent biomarker for predicting the therapeutic efficacy of GC1118 in GBM tumors. In summary, GC1118 exerted a potent anti-tumor effect on GBM tumors in PDX models, and its therapeutic efficacy was especially pronounced in the tumors with high EGFR amplification. Our study supports the importance of patient stratification based on EGFR copy number variation in clinical trials for GBM. The superiority of GC1118 over other EGFR mAbs in GBM tumors should be assessed in future studies.
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Diffusely infiltrating gliomas (DIGs) are difficult to completely resect and are associated with a high rate of tumor relapse and progression from low- to high-grade glioma. In particular, optimized short-term culture-enriching patient-derived glioma stem cells (GSCs) are essential for customizing the therapeutic strategy based on clinically feasible in vitro drug screening for a wide range of DIGs, owing to the high inter-tumoral heterogeneity. Herein, we constructed a novel high-throughput culture condition screening platform called 'GFSCAN', which evaluated the cellular growth rates of GSCs for each DIG sample in 132 serum-free combinations, using 13 previously reported growth factors closely associated with glioma aggressiveness. In total, 72 patient-derived GSCs with available genomic profiles were tested in GFSCAN to explore the association between cellular growth rates in specific growth factor combinations and genomic/molecular backgrounds, including isocitrate dehydrogenase 1 (IDH1) mutation, chromosome arm 1p and 19q co-deletion, ATRX chromatin remodeler alteration, and transcriptional subtype. GSCs were clustered according to the dependency on epidermal growth factor and basic fibroblast growth factor (E&F), and isocitrate dehydrogenase 1 (IDH1) wild-type GSCs showed higher E&F dependencies than IDH1 mutant GSCs. More importantly, we elucidated optimal combinations for IDH1 mutant glioblastoma and lower grade glioma GSCs with low dependencies on E&F, which could be an aid in clinical decision-making for these DIGs. Thus, we demonstrated the utility of GFSCAN in personalizing in vitro cultivation to nominate personalized therapeutic options, in a clinically relevant time frame, for individual DIG patients, where standard clinical options have been exhausted.
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BACKGROUND: Glioblastoma (GBM) is a complex disease with extensive molecular and transcriptional heterogeneity. GBM can be subcategorized into four distinct subtypes; tumors that shift towards the mesenchymal phenotype upon recurrence are generally associated with treatment resistance, unfavorable prognosis, and the infiltration of pro-tumorigenic macrophages. RESULTS: We explore the transcriptional regulatory networks of mesenchymal-associated tumor-associated macrophages (MA-TAMs), which drive the malignant phenotypic state of GBM, and identify macrophage receptor with collagenous structure (MARCO) as the most highly differentially expressed gene. MARCOhigh TAMs induce a phenotypic shift towards mesenchymal cellular state of glioma stem cells, promoting both invasive and proliferative activities, as well as therapeutic resistance to irradiation. MARCOhigh TAMs also significantly accelerate tumor engraftment and growth in vivo. Moreover, both MA-TAM master regulators and their target genes are significantly correlated with poor clinical outcomes and are often associated with genomic aberrations in neurofibromin 1 (NF1) and phosphoinositide 3-kinases/mammalian target of rapamycin/Akt pathway (PI3K-mTOR-AKT)-related genes. We further demonstrate the origination of MA-TAMs from peripheral blood, as well as their potential association with tumor-induced polarization states and immunosuppressive environments. CONCLUSIONS: Collectively, our study characterizes the global transcriptional profile of TAMs driving mesenchymal GBM pathogenesis, providing potential therapeutic targets for improving the effectiveness of GBM immunotherapy.
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Redes Reguladoras de Genes , Glioblastoma/genética , Macrófagos Associados a Tumor , Animais , Carcinogênese , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioma/genética , Humanos , Imunoterapia , Macrófagos/metabolismo , Camundongos , Neurofibromina 1/genética , Fenótipo , Prognóstico , Células-Tronco , Transcriptoma , Microambiente TumoralRESUMO
As glioblastomas are mostly localized infiltrative lesions, gene therapy based on the retroviral replicating vector (RRV) system is considered an attractive strategy. Combinations of multiple suicide genes can circumvent the limitations associated with each gene, achieving direct and synergistic cytotoxic effects, along with bystander cell killing. In this study, we constructed a semi-and pseudotyped-RRV (sp-RRV) system harboring two suicide genes-herpes simplex virus type 1 thymidine kinase (TK) and yeast cytosine deaminase (CD)-to verify the dissemination and antitumor efficacy of our sp-RRV system (spRRVe-sEF1α-TK/sRRVgp-sEF1α-CD) in seven patient-derived glioblastoma stem-like cells (GSCs). Flow cytometry and high-content analysis revealed a wide range of transduction efficiency and good correlation between the delivery of therapeutic genes and susceptibility to the prodrugs ganciclovir and 5-fluorocytosine in patient-derived GSCs in vitro. Intra-tumoral delivery of spRRVe-sEF1α-TK/sRRVgp-sEF1α-CD, combined with prodrug treatment, synergistically inhibited cell proliferation and angiogenesis while increasing apoptosis and the depletion of tumor-associated macrophages in orthotopic glioblastoma xenografts. Genomic profiling of patient-derived GSCs revealed that the key genes preventing sp-RRV infection and transmission were associated with cell adhesion, migration, development, differentiation, and proliferation. This is the first report demonstrating that a novel sp-RRV-mediated TK/CD double suicide gene transfer system has high oncolytic power against extremely heterogeneous and treatment-refractory glioblastomas.
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Glioblastoma (GBM) is the most lethal primary brain tumor with few treatment options. The survival of glioma-initiating cells (GICs) is one of the major factors contributing to treatment failure. GICs frequently produce and respond to their own growth factors that support cell proliferation and survival. In this study, we aimed to identify critical autocrine factors mediating GIC survival and to evaluate the anti-GBM effect of antagonizing these factors. Proteomic analysis was performed using conditioned media from two different patient-derived GBM tumor spheres under a growth factor-depleted status. Then, the antitumor effects of inhibiting an identified autocrine factor were evaluated by bioinformatic analysis and molecular validation. Proteins secreted by sphere-forming GICs promote cell proliferation/survival and detoxify reactive oxygen species (ROS). Among these proteins, we focused on midkine (MDK) as a clinically significant and pathologically relevant autocrine factor. Antagonizing MDK reduced the survival of GBM tumor spheres through the promotion of cell cycle arrest and the consequent apoptotic cell death caused by oxidative stress-induced DNA damage. We also identified PCBP4, a novel molecular predictor of resistance to anti-MDK treatment. Collectively, our results indicate that MDK inhibition is an important therapeutic option by suppressing GIC survival through the induction of ROS-mediated cell cycle arrest and apoptosis.
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Sistema Nervoso Central/metabolismo , Glioblastoma/metabolismo , Midkina/metabolismo , Proteínas de Ligação a RNA/metabolismo , Apoptose/genética , Apoptose/fisiologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Biologia Computacional , Dano ao DNA/genética , Dano ao DNA/fisiologia , Humanos , Técnicas In Vitro , Espécies Reativas de Oxigênio/metabolismo , Análise de Sequência de RNARESUMO
BACKGROUND: Cancer is a complex disease with profound genomic alterations and extensive heterogeneity. Recent studies on large-scale genomics have shed light on the impact of core oncogenic pathways, which are frequently dysregulated in a wide spectrum of cancer types. Aberrant activation of the hepatocyte growth factor (HGF) signaling axis has been associated with promoting various oncogenic programs during tumor initiation, progression, and treatment resistance. As a result, HGF-targeted therapy has emerged as an attractive therapeutic approach. However, recent clinical trials involving HGF-targeted therapies have demonstrated rather disappointing results. Thus, an alternative, in-depth assessment of new patient stratification is necessary to shift the current clinical course. METHODS: To address such challenges, we have evaluated the therapeutic efficacy of YYB-101, an HGF-neutralizing antibody, in a series of primary glioblastoma stem cells (GSCs) both in vitro and in vivo. Furthermore, we performed genome and transcriptome analysis to determine genetic and molecular traits that exhibit therapeutic susceptibility to HGF-mediated therapy. RESULTS: We have identified several differentially expressed genes, including MET, KDR, and SOX3, which are associated with tumor invasiveness, malignancy, and unfavorable prognosis in glioblastoma patients. We also demonstrated the HGF-MET signaling axis as a key molecular determinant in GSC invasion, and we discovered that a significant association in HGF expression existed between mesenchymal phenotype and immune cell recruitment. CONCLUSIONS: Upregulation of MET and mesenchymal cellular state are essential in generating HGF-mediated therapeutic responses. Our results provide an important framework for evaluating HGF-targeted therapy in future clinical settings.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genômica/métodos , Glioblastoma/tratamento farmacológico , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Transcriptoma , Animais , Apoptose , Movimento Celular , Proliferação de Células , Feminino , Glioblastoma/genética , Glioblastoma/patologia , Fator de Crescimento de Hepatócito/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fenótipo , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Glioblastoma (GBM) is the most malignant brain tumor with profound genomic alterations. Tumor suppressor genes regulate multiple signaling networks that restrict cellular proliferation and present barriers to malignant transformation. While bona fide tumor suppressors such as PTEN and TP53 often undergo inactivation due to mutations, there are several genes for which genomic deletion is the primary route for tumor progression. To functionally identify putative tumor suppressors in GBM, we employed in vivo RNAi screening using patient-derived xenograft models. Here, we identified PIP4K2A, whose functional role and clinical relevance remain unexplored in GBM. We discovered that PIP4K2A negatively regulates phosphoinositide 3-kinase (PI3K) signaling via p85/p110 component degradation in PTEN-deficient GBMs and specifically targets p85 for proteasome-mediated degradation. Overexpression of PIP4K2A suppressed cellular and clonogenic growth in vitro and impeded tumor growth in vivo. Our results unravel a novel tumor-suppressive role of PIP4K2A for the first time and support the feasibility of combining oncogenomics with in vivo RNAi screen.
Assuntos
Neoplasias Encefálicas/metabolismo , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Glioblastoma/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Neoplasias Encefálicas/patologia , Carcinogênese/metabolismo , Proliferação de Células/genética , Células Cultivadas , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Feminino , Glioblastoma/patologia , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Interferência de RNA , Transdução Genética , Carga Tumoral/genéticaRESUMO
Background: Despite extensive efforts on the genomic characterization of gliomas, very few studies have reported the genetic alterations of cerebellar glioblastoma (C-GBM), a rare and lethal disease. Here, we provide a systematic study of C-GBM to better understand its specific genomic features. Methods: We collected a cohort of C-GBM patients and compared patient demographics and tumor pathologies with supratentorial glioblastoma (S-GBM). To uncover the molecular characteristics, we performed DNA and mRNA sequencing and DNA methylation arrays on 19, 6, and 4 C-GBM cases, respectively. Moreover, chemical drug screening was conducted to identify potential therapeutic options for C-GBMs. Results: Despite differing anatomical origins of C-GBM and S-GBM, neither histological, cytological, nor patient demographics appeared significantly different between the 2 types. However, we observed striking differences in mutational patterns, including frequent alterations of ATRX, PDGFRA, NF1, and RAS and absence of EGFR alterations in C-GBM. These results show a distinct evolutionary path in C-GBM, suggesting specific therapeutic targeted options. Targeted-drug screening revealed that C-GBMs were more responsive to mitogen-activated protein kinase kinase (MEK) inhibitor and resistant to epidermal growth factor receptor inhibitors than S-GBMs. Also, differential expression analysis indicated that C-GBMs may have originated from oligodendrocyte progenitor cells, suggesting that different types of cells can undergo malignant transformation according to their location in brain. Master regulator analysis with differentially expressed genes between C-GBM and proneural S-GBM revealed NR4A1 as a potential therapeutic target. Conclusions: Our results imply that unique gliomagenesis mechanisms occur in adult cerebellum and new treatment strategies are needed to provide greater therapeutic benefits for C-GBM patients. Key Points: 1. Distinct genomic profiles of 19 adult cerebellar GBMs were characterized. 2. MEK inhibitor was highly sensitive to cerebellar GBM compared with supratentorial GBM. 3. Master regulator analysis revealed NR4A1 as a potential therapeutic target in cerebellar GBM.