RESUMEN
In xenograft models, orthotopic (ORT) engraftment is thought to provide a different tumor microenvironment compared with subcutaneous (SC) engraftment. We attempted to characterize the biological difference between OE19 (adenocarcinoma of the gastroesophageal junction) SC and ORT models by pathological analysis and CASTIN (CAncer-STromal INteractome) analysis, which is a novel method developed to analyze the tumor-stroma interactome framework. In SC models, SCID mice were inoculated subcutaneously with OE19 cells, and tumor tissues were sampled at 3 weeks. In ORT models, SCID mice were inoculated under the serosal membrane of the stomach wall, and tumor tissues were sampled at 3 and 6 weeks after engraftment. Results from the two models were then compared. Histopathologically, the SC tumors were well circumscribed from the adjacent tissue, with scant stroma and the formation of large ductal structures. In contrast, the ORT tumors were less circumscribed, with small ductal structures invading into abundant stroma. Then we compared the transcriptome profiles of human tumor cells with the mouse stromal cells of each model by species-specific RNA sequencing. With CASTIN analysis, we successfully identified several interactions that are known to affect the tumor microenvironment as being selectively enhanced in the ORT model. In conclusion, pathological analysis and CASTIN analysis revealed that ORT models of OE19 cells have a more invasive character and enhanced interaction with stromal cells compared with SC models.
RESUMEN
It is ideal for the target antigen of a cytotoxic therapeutic antibody against cancer to be cancer-specific, but such antigens are rare. Thus an alternative strategy for target selection is necessary. Desmoglein 3 (DSG3) is highly expressed in lung squamous cell carcinoma, while it is well-known that anti-DSG3 antibodies cause pemphigus vulgaris, an autoimmune disease. We evaluated DSG3 as a novel target by selecting an epitope that exerts efficacy against cancer with no pathogenic effects in normal tissues. Pathogenic anti-DSG3 antibodies induce skin blisters by inhibiting the cell-cell interaction in a Ca2+-dependent manner. We screened anti-DSG3 antibodies that bind DGS3 independent of Ca2+ and have high antibody-dependent cell cytotoxicity (ADCC) activity against DSG3-expressing cells. These selected antibodies did not inhibit cell-cell interaction and showed ADCC activity against squamous cell carcinoma cell lines. Furthermore, one of the DSG3 antibodies showed anti-tumour activity in tumour mouse models but did not induce adverse effects such as blister formation in the skin. Thus it was possible to generate an antibody against DSG3 by using an appropriate epitope that retained efficacy with no pathogenicity. This approach of epitope selection may expand the variety of druggable target molecules.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Desmogleína 3/antagonistas & inhibidores , Diseño de Fármacos , Queratinocitos/efectos de los fármacos , Animales , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/farmacología , Especificidad de Anticuerpos , Antineoplásicos Inmunológicos/efectos adversos , Antineoplásicos Inmunológicos/farmacología , Células CHO , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cricetulus , Desmogleína 3/química , Desmogleína 3/genética , Desmogleína 3/metabolismo , Epítopos/química , Epítopos/genética , Epítopos/metabolismo , Humanos , Queratinocitos/inmunología , Queratinocitos/metabolismo , Queratinocitos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
RHOA missense mutations exist specifically in diffuse type gastric cancers (DGC) and are considered one of the DGC driver genes, but it is not fully understood how RHOA mutations contribute to DGC development. Here we examined how RHOA mutations affect cancer cell survival and cell motility. We revealed that cell survival was maintained by specific mutation sites, namely G17, Y42, and L57. Because these functional mutations suppressed MLC2 phosphorylation and actin stress fiber formation, we realized they act in a dominant-negative fashion against the ROCK pathway. Through the same inactivating mechanism that maintained cell survival, RHOA mutations also increased cell migration activity. Cell survival and migration studies on CLDN18-ARHGAP (CLG) fusions, which are known to be mutually exclusive to RHOA mutations, showed that CLG fusions complemented cell survival under RHOA knockdown condition and also induced cell migration. Site-directed mutagenesis analysis revealed the importance of the GAP domain and indicated that CLG fusions maintained RHOA in the inactive form. Taken together, these findings show that the inactivation of ROCK would be a key step in DGC development, so ROCK activation might provide novel therapeutic opportunities.
RESUMEN
Leucine-rich repeat-containing G protein-coupled receptor 6 (LGR6) is a seven-pass transmembrane protein known to be a marker of stem cells in several organs. To deepen our understanding of the cell biology of LGR6-positive cells, including stem cells, we generated monoclonal antibodies (mAbs) against human LGR6. DNA immunization followed by whole-cell immunization with LGR6-expressing transfectants was performed to obtain mAbs that recognized the native form of LGR6. Hybridomas were screened by flow cytometry using LGR6-transfected cells. Because the molecules of LGR4, LGR5, and LGR6 are 50% homologous at the amino acid level, specificity of the mAbs was confirmed by transfectants expressing LGR4, LGR5, or LGR6. Three LGR6-specific mAbs were generated. Two of the three mAbs (designated 43A6 and 43D10) recognized the large N-terminal extracellular domain of LGR6, and competitively blocked the binding of R-spondin 1, which is known to be the ligand for LGR6. The other mAb, 43A25, recognized the seven-pass transmembrane domain of LGR6, and was able to be used for immunoblot analysis. In addition, mAbs 43A6 and 43D10 detected endogenous expression of LGR6 in cancer cell lines. We expect that our mAbs will contribute to widening our understanding of LGR6-positive cells in humans.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Receptores Acoplados a Proteínas G/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/metabolismo , Unión Competitiva , Línea Celular , Línea Celular Tumoral , Citometría de Flujo , Humanos , Hibridomas , Immunoblotting , Ratones Endogámicos BALB C , Unión Proteica , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/metabolismoRESUMEN
BACKGROUND: Knockdown or overexpression of genes is widely used to identify genes that play important roles in many aspects of cellular functions and phenotypes. Because next-generation sequencing generates high-throughput data that allow us to detect genes, it is important to identify genes that drive functional and phenotypic changes of cells. However, conventional methods rely heavily on the assumption of normality and they often give incorrect results when the assumption is not true. To relax the Gaussian assumption in causal inference, we introduce the non-paranormal method to test conditional independence in the PC-algorithm. Then, we present the non-paranormal intervention-calculus when the directed acyclic graph (DAG) is absent (NPN-IDA), which incorporates the cumulative nature of effects through a cascaded pathway via causal inference for ranking causal genes against a phenotype with the non-paranormal method for estimating DAGs. RESULTS: We demonstrate that causal inference with the non-paranormal method significantly improves the performance in estimating DAGs on synthetic data in comparison with the original PC-algorithm. Moreover, we show that NPN-IDA outperforms the conventional methods in exploring regulators of the flowering time in Arabidopsis thaliana and regulators that control the browning of white adipocytes in mice. Our results show that performance improvement in estimating DAGs contributes to an accurate estimation of causal effects. CONCLUSIONS: Although the simplest alternative procedure was used, our proposed method enables us to design efficient intervention experiments and can be applied to a wide range of research purposes, including drug discovery, because of its generality.
Asunto(s)
Algoritmos , Técnicas Genéticas , Adipocitos Marrones/citología , Adipocitos Marrones/metabolismo , Adipocitos Blancos/citología , Adipocitos Blancos/metabolismo , Animales , Arabidopsis/genética , Interpretación Estadística de Datos , Técnicas de Silenciamiento del Gen , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Distribución Normal , Fenotipo , Análisis de RegresiónRESUMEN
We have been investigating the functional display of multipass membrane protein such as transporter or G-protein coupled receptor on the budded baculovirus (BV). We tested the use of a viral envelope protein gp64 transgenic mouse for the direct immunization of these membrane proteins displayed on BVs. The gp64 transgenic mice showed only a weak response to virus compared to wild type BALB/c mice. Immunizing gp64 transgenic mice with the BV expressing peptide transporter PepT1, we obtained 47 monoclonal antibodies (mAbs). These mAbs were specific to the PepT1 on the pancreatic cancer cells AsPC-1 by fluorocytometric analysis and exhibited antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity to AsPC-1. We also generated 7 mAbs by immunizing gp64 transgenic mice on a CCR2-deficient background with the BV expressing chemokine receptor CCR2 together with partially purified CCR2. These mAbs possessed specific binding to CCR2 in CHO cells on fluorocytometric analysis, and exhibited neutralizing activities for ligand-dependent inhibition of cyclic AMP production. This method provides a powerful tool for the generation of therapeutic/diagnostic mAbs against membrane proteins.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Baculoviridae/genética , Moléculas de Adhesión Celular/genética , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/inmunología , Biblioteca de Péptidos , Proteínas del Envoltorio Viral/genética , Proteínas Virales/genética , Animales , Baculoviridae/metabolismo , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Inmunización , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Transportador de Péptidos 1 , Receptores CCR2 , Receptores de Quimiocina/inmunología , Simportadores/inmunología , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
PURPOSE: Hepatocellular carcinoma is the most common primary malignancy of the liver and accounts for as many as one million deaths annually worldwide. The present study was done to identify new transmembrane molecules for antibody therapy in hepatocellular carcinoma. EXPERIMENTAL DESIGN: Gene expression profiles of pooled total RNA from three tissues each of moderately differentiated and poorly differentiated hepatocellular carcinoma were compared with those of normal liver, noncancerous liver tissue in hepatocellular carcinoma patients, 30 normal tissue samples, and five fetal tissue samples. Target genes up-regulated specifically in hepatocellular carcinoma were validated by immunohistochemical analysis and complement-dependent cytotoxicity assay using monoclonal antibodies generated against target molecules. RESULTS: The human homologue of the Drosophila Roundabout gene, axon guidance receptor homologue 1, ROBO1/DUTT1, a member of the immunoglobulin superfamily, was highly expressed in hepatocellular carcinoma, whereas it showed only a limited distribution in normal tissues. On immunohistochemical analysis using a newly generated anti-ROBO1 monoclonal antibody, positive signals were observed in 83 of 98 cases of hepatocellular carcinoma (84.7%). The mAb B2318C induced complement-dependent cytotoxicity in ROBO1-expressing cell lines and in the liver cancer cell line PLC/PRF/5. Strikingly, the ectodomain of ROBO1 was detected not only in the culture medium of liver cancer cell lines (PLC/PRF/5, HepG2, etc.) but also in sera from hepatocellular carcinoma patients (6 of 11). CONCLUSIONS: This is the first report that ROBO1 is overexpressed in hepatocellular carcinoma and shed into serum in humans. These observations suggest that ROBO1 is a potential new serologic marker for hepatocellular carcinoma and may represent a new therapeutic target.
Asunto(s)
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteínas del Tejido Nervioso/genética , Receptores Inmunológicos/genética , Animales , Anticuerpos Monoclonales/farmacología , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Células COS , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/diagnóstico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Chlorocebus aethiops , Pruebas Inmunológicas de Citotoxicidad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/diagnóstico , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/sangre , Estructura Terciaria de Proteína , ARN Mensajero/biosíntesis , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Regulación hacia Arriba/genética , Proteínas RoundaboutRESUMEN
Transporters play a critical role in many physiological and pathological states and expression of the functional transporter protein is essential in exploring its kinetics and developing effective drugs. We describe here the recovery of functional transporter protein in the baculovirus fraction. We introduced a gene encoding human peptide transporter PepT1, important for the absorption of protein hydrolytic products or peptide-mimetic drugs, into a baculovirus vector. After infection, a large amount of PepT1 appeared in the budded virus fraction compared with Sf9 cells. Uptake of [14C]glycylsarcosine was markedly increased in an acidic condition and showed a clear overshoot in PepT1-expressing virus fraction. The apparent Michaelis constant for [14C]glycylsarcosine was 0.55 +/- 0.06 mM. [14C]Glycylsarcosine uptake was inhibited by di- and tripeptides and orally active beta-lactam antibiotics. These results suggest that functional PepT1 recovers efficiently in a budded virus fraction, and, thus, this expression system will be a useful tool for characterization and screening of peptide-mimetic drugs in drug discovery.