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Glioblastoma is characterized by diffuse infiltration into the normal brain. Invasive glioma stem cells (GSCs) are an underlying cause of treatment failure. Despite the use of multimodal therapies, the prognosis remains dismal. New therapeutic approach targeting invasive GSCs is required. Here, we show that neural stem cells (NSCs) derived from CRISRP/Cas9-edited human-induced pluripotent stem cell (hiPSC) expressing a suicide gene had higher tumor-trophic migratory capacity compared with mesenchymal stem cells (MSCs), leading to marked in vivo antitumor effects. High migratory capacity in iPSC-NSCs was related to self-repulsive action and pathotropism involved in EphB-ephrinB and CXCL12-CXCR4 signaling. The gene insertion to ACTB provided higher and stable transgene expression than other common insertion sites, such as GAPDH or AAVS1. Ferroptosis was associated with enhanced antitumor immune responses. The thymidylate synthase and dihydroprimidine dehydrogenase expressions predicted the treatment efficacy of therapeutic hiPSC-NSCs. Our results indicate the potential benefit of genome-edited iPS cells based gene therapy for invasive GSCs. Furthermore, the present research concept may become a platform to promote clinical studies using hiPSC.
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Experimental techniques for patient-derived cancer stem-cell organoids/spheroids can be powerful diagnostic tools for personalized chemotherapy. However, establishing their cultures from gastric cancer remains challenging due to low culture efficiency and cumbersome methods. To propagate gastric cancer cells as highly proliferative stem-cell spheroids in vitro, we initially used a similar method to that for colorectal cancer stem cells, which, unfortunately, resulted in a low success rate (25%, 18 of 71 cases). We scrutinized the protocol and found that the unsuccessful cases were largely caused by the paucity of cancer stem cells in the sampled tissues as well as insufficient culture media. To overcome these obstacles, we extensively revised our sample collection protocol and culture conditions. We then investigated the following second cohort and, consequently, achieved a significantly higher success rate (88%, 29 of 33 cases). One of the key improvements included new sampling procedures for tumor tissues from wider and deeper areas of gastric cancer specimens, which allowed securing cancer stem cells more reproducibly. Additionally, we embedded tumor epithelial pieces separately in both Matrigel and collagen type-I as their preference to the extracellular matrix was different depending on the tumors. We also added a low concentration of Wnt ligands to the culture, which helped the growth of occasional Wnt-responsive gastric cancer stem-cell spheroids without allowing proliferation of the normal gastric epithelial stem cells. This newly improved spheroid culture method may facilitate further studies, including personalized drug-sensitivity tests prior to drug therapy.
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Esferoides Celulares , Neoplasias Gástricas , Humanos , Esferoides Celulares/patologia , Neoplasias Gástricas/patologia , Células-Tronco Neoplásicas/patologiaRESUMO
Recent advances in combinatorial chemistry led to the discovery of inhibitors targeting the KRAS G12C-mutant protein. However, efficacy of its monotherapy on colorectal cancer is limited. Thus, effective combination drugs should be explored for applicable patients with colorectal cancer to fully benefit from the KRAS G12C inhibitor treatment. Here we used a patient-derived colorectal cancer stem cell (PD-CRC-SC) spheroid culture and showed that three-drug combination of inhibitors against KRAS G12C, EGFR, and FGFR synergistically suppressed the growth of colorectal cancer cells carrying the KRAS G12C mutation. Likewise, a combination of KRAS G12C and SHP2 inhibitors was also effective. Importantly, activation of the PI3K/AKT pathway in heregulin-responsive colorectal cancer cells canceled out the effect of KRAS G12C inhibition, which was largely overcome by PI3K inhibitors. These results reveal that evaluating efficacy of combination therapies with PD-CRC-SC spheroids can be a promising strategy to find the best regimen for patients with colorectal cancer.
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Neoplasias Colorretais , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismoRESUMO
Reprogramming of energy metabolism is regarded as one of the hallmarks of cancer; in particular, oncogenic RAS has been shown to be a critical regulator of cancer metabolism. Recently, asparagine metabolism has been heavily investigated as a novel target for cancer treatment. For example, Knott et al. showed that asparagine bioavailability governs metastasis in a breast cancer model. Gwinn et al. reported the therapeutic vulnerability of asparagine biosynthesis in KRAS-driven non-small cell lung cancer. We previously reported that KRAS-mutated CRC cells can adapt to glutamine depletion through upregulation of asparagine synthetase (ASNS), an enzyme that synthesizes asparagine from aspartate. In our previous study, we assessed the efficacy of asparagine depletion using human cancer cell lines. In the present study, we evaluated the clinical relevance of asparagine depletion using a novel patient-derived spheroid xenograft (PDSX) mouse model. First, we examined ASNS expression in 38 spheroid lines and found that 12 lines (12/37, 32.4%) displayed high ASNS expression, whereas 26 lines (25/37, 67.6%) showed no ASNS expression. Next, to determine the role of asparagine metabolism in tumor growth, we established ASNS-knockdown spheroid lines using lentiviral short hairpin RNA constructs targeting ASNS. An in vitro cell proliferation assay demonstrated a significant decrease in cell proliferation upon asparagine depletion in the ASNS-knockdown spheroid lines, and this was not observed in the control spheroids lines. In addition, we examined asparagine inhibition with the anti-leukemia drug L-asparaginase (L-Asp) and observed a considerable reduction in cell proliferation at a low concentration (0.1 U/mL) in the ASNS-knockdown spheroid lines, whereas it exhibited limited inhibition of control spheroid lines at the same concentration. Finally, we used the PDSX model to assess the effects of asparagine depletion on tumor growth in vivo. The nude mice injected with ASNS-knockdown or control spheroid lines were administered with L-Asp once a day for 28 days. Surprisingly, in mice injected with ASNS-knockdown spheroids, the administration of L-Asp dramatically inhibited tumor engraftment. On the other hands, in mice injected with control spheroids, the administration of L-Asp had no effect on tumor growth inhibition at all. These results suggest that ASNS inhibition could be critical in targeting asparagine metabolism in cancers.
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Aspartato-Amônia Ligase , Carcinogênese , Animais , Humanos , Camundongos , Asparaginase/farmacologia , Asparaginase/metabolismo , Asparagina/metabolismo , Aspartato-Amônia Ligase/genética , Aspartato-Amônia Ligase/metabolismo , Ácido Aspártico , Carcinoma Pulmonar de Células não Pequenas , Linhagem Celular Tumoral , Glutamina , Neoplasias Pulmonares , Camundongos Nus , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Interferente Pequeno , Carcinogênese/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Esferoides CelularesRESUMO
Mutations of KRAS gene are found in various types of cancer, including colorectal cancer (CRC). Despite intense efforts, no pharmacological approaches are expected to be effective against KRAS-mutant cancers. Macropinocytosis is an evolutionarily conserved actin-dependent endocytic process that internalizes extracellular fluids into large vesicles called macropinosomes. Recent studies have revealed macropinocytosis's important role in metabolic adaptation to nutrient stress in cancer cells harboring KRAS mutations. Here we showed that KRAS-mutant CRC cells enhanced macropinocytosis for tumor growth under nutrient-depleted conditions. We also demonstrated that activation of Rac1 and phosphoinositide 3-kinase were involved in macropinocytosis of KRAS-mutant CRC cells. Furthermore, we found that macropinocytosis was closely correlated with asparagine metabolism. In KRAS-mutant CRC cells engineered with knockdown of asparagine synthetase, macropinocytosis was accelerated under glutamine-depleted condition, and albumin addition could restore the glutamine depletion-induced growth suppression by recovering the intracellular asparagine level. Finally, we discovered that the combination of macropinocytosis inhibition and asparagine depletion dramatically suppressed the tumor growth of KRAS-mutant CRC cells in vivo. These results indicate that dual blockade of macropinocytosis and asparagine bioavailability could be a novel therapeutic strategy for KRAS-mutant cancers.
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Aspartato-Amônia Ligase/genética , Neoplasias Colorretais/terapia , Pinocitose/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Asparagina/genética , Asparagina/metabolismo , Aspartato-Amônia Ligase/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Técnicas de Silenciamento de Genes , Humanos , Mutação/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Tumor-infiltrating lymphocytes (TIL), which include tumor-specific T lymphocytes with frequency, are used for adoptive cell transfer therapy (ACT) in clinical practice. The optimization of TIL preparation has been investigated to reduce the senescence and increase the abundance of TIL, as both the quality and quantity of the transferred cells have great influence on the outcome of TIL-based ACT (TIL-ACT). Considering the effects of cell reprogramming on senescence, we expected that the anti-tumor effect could be enhanced by TIL regeneration. To confirm this hypothesis, we established tumor-specific TIL-derived iPS cells (TIL-iPSC) with human colorectal cancer specimens. T cells differentiated from TIL-iPSC (TIL-iPS-T) retained not only intrinsic T cell functions and tumor specificity, but also exhibited improved proliferation capacity and additional killing activity. Moreover, less differentiated profiles and prolonged persistency were seen in TIL-iPS-T compared with primary cells. Our findings imply that iPSC technology has great potential for TIL-ACT.
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Neoplasias Colorretais/terapia , Células-Tronco Pluripotentes Induzidas/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Neoplasias Colorretais/imunologia , Feminino , Humanos , Imunoterapia , Células-Tronco Pluripotentes Induzidas/citologia , Ativação Linfocitária , Linfócitos do Interstício Tumoral/citologia , Camundongos Endogâmicos NOD , Camundongos SCID , Linfócitos T/citologia , Linfócitos T/transplanteRESUMO
Humans with biallelic inactivating mutations in Epithelial Cell Adhesion Molecule (EpCAM) develop congenital tufting enteropathy (CTE). To gain mechanistic insights regarding EpCAM function in this disorder, we prepared intestinal epithelial cell (IEC) organoids and spheroids. IEC organoids and spheroids were generated from ROSA-CreERT2 EpCAMfl/fl mice. Proliferation, tight junctions, cell polarity and epithelial integrity were assessed in tamoxifen-induced EpCAM-deficient organoids via confocal immunofluorescence microscopy and Western blotting. Olfm4-expressing stem cells were assessed in IEC cells in vitro and in vivo via fluorescence in situ hybridization. To determine if existing drugs could ameliorate effects of EpCAM deficiency in IEC cells, a variety of pharmacologic inhibitors were screened. Deletion of EpCAM resulted in increased apoptosis and attenuated growth of organoids and spheroids. Selected claudins were destabilized and epithelial integrity was severely compromised. Epithelial integrity was improved by treatment with Rho-associated coiled-coil kinase (ROCK) inhibitors without restoration of claudin expression. Correspondingly, enhanced phosphorylation of myosin light chain, a serine/threonine ROCK substrate, was observed in EpCAM-deficient organoids. Strikingly, frequencies of Olfm4-expressing stem cells in EpCAM-deficient IEC cells in vitro and in vivo were decreased. Treatment with ROCK inhibitors increased numbers of stem cells in EpCAM-deficient organoids and spheroids. Thus, EpCAM regulates intestinal epithelial homeostasis via a signaling pathway that includes ROCK.
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Molécula de Adesão da Célula Epitelial/metabolismo , Células Epiteliais/metabolismo , Intestinos/citologia , Células-Tronco/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Claudinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Inativação Gênica , Mucosa Intestinal/metabolismo , Camundongos Knockout , Cadeias Leves de Miosina/metabolismo , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/ultraestrutura , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Células-Tronco/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Objective: We report a rare case of a ruptured anterior spinal artery (ASA) aneurysm caused by bilateral vertebral artery (VA) occlusion. Case Presentations: A 78-year-old man suddenly developed severe headache and slight hemiparesis, and was admitted to our hospital. Computed tomography (CT) revealed subarachnoid hemorrhage, mainly in the posterior fossa. On emergency angiography, the right VA terminated at the origin of the posterior inferior cerebellar artery (PICA), and anastomoses between the PICA and the anterior inferior cerebellar artery (AICA) were observed, in addition to a saccular 3-mm aneurysm with bleb originating from the PICA-AICA anastomosis. Left vertebral arteriography demonstrated that the left VA was occluded segmentally at the V4 level and revealed a tortuous arterial network filling the distal VA. Based on the location of the bleeding, the right VA aneurysm was considered to have ruptured. After balloon test occlusion of the right VA, parent artery occlusion was performed without complications. The patient had no neurological changes immediately after surgery, but several hours later, he stopped breathing. Retrospective analysis revealed an ASA aneurysm, which was determined to be the bleeding source. Although conservative treatment was performed, he died the fourth day after onset without neurological improvement. Conclusion: In cases of subarachnoid hemorrhage associated with bilateral VA occlusion, an aneurysm formed by hemodynamic stress may be the source of hemorrhage. It is important to suspect aneurysms in the extracranial collaterals, such as the ASA, and intracranial collaterals such as the PICA-AICA anastomosis.
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Malignant glioma, which is characterized by diffuse infiltration into the normal brain parenchyma, is the most aggressive primary brain tumor with dismal prognosis. Over the past 40 years, the median survival has only slightly improved. Therefore, new therapeutic modalities must be developed. In the 1990s, suicide gene therapy began attracting attention for the treatment of malignant glioma. Some clinical trials used a viral vector for suicide gene transduction; however, it was found that viral vectors cannot cover the large invaded area of glioma cells. Interest in this therapy was recently revived because some types of stem cells possess a tumor-tropic migratory capacity, which can be used as cellular delivery vehicles. Immortalized, clonal neural stem cell (NSC) line has been used for patients with recurrent high-grade glioma, which showed safety and efficacy. Embryonic and induced pluripotent stem cells may be considered as sources of NSC because NSC is difficult to harvest, and ethical issues have been raised. Mesenchymal stem cells are alternative candidates for cellular vehicle and are easily harvested from the bone marrow. In addition, a new type of nonlytic, amphotropic retroviral replicating vector encoding suicide gene has shown efficacy in patients with recurrent high-grade glioma in a clinical trial. This replicating viral capacity is another possible candidate as delivery vehicle to tackle gliomas. Herein, we review the concept of suicide gene therapy, as well as recent progress in preclinical and clinical studies in this field.
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Neoplasias Encefálicas/terapia , Genes Transgênicos Suicidas/genética , Terapia Genética/métodos , Glioma/terapia , Ensaios Clínicos como Assunto , HumanosRESUMO
Some colorectal cancer patients harboring FGFR (fibroblast growth factor receptor) genetic alterations, such as copy number gain, mutation, and/or mRNA overexpression, were selected for enrollment in several recent clinical trials of FGFR inhibitor, because these genetic alterations were preclinically reported to be associated with FGFR inhibitor sensitivity as well as poor prognosis, invasiveness, and/or metastatic potential. However, few enrolled patients were responsive to FGFR inhibitors. Thus, practical strategies are eagerly awaited that can stratify patients for the subset that potentially responds to FGFR inhibitor chemotherapy. In the present study, we evaluated the sensitivity to FGFR inhibitor erdafitinib on 25 patient-derived tumor-initiating cell (TIC) spheroid lines carrying wild-type RAS and RAF genes, both in vitro and in vivo. Then, we assessed possible correlations between the sensitivity and the genetic/genomic data of the spheroid lines tested. Upon their exposure to erdafitinib, seven lines (7/25, 28%) responded significantly. Normal colonic epithelial stem cells were unaffected by the inhibitors. Moreover, the combination of erdafitinib with EGFR inhibitor erlotinib showed stronger growth inhibition than either drug alone, as efficacy was observed in 21 lines (84%) including 14 (56%) that were insensitive to erdafitinib alone. The in vitro erdafitinib response was accurately reflected on mouse xenografts of TIC spheroid lines. However, we found little correlation between their genetic/genomic alterations of TIC spheroids and the sensitivity to the FGFR inhibitor. Accordingly, we propose that direct testing of the patient-derived spheroids in vitro is one of the most reliable personalized methods in FGFR-inhibitor therapy of colorectal cancer patients.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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The Jun dimerization protein 2 (Jdp2) is expressed predominantly in granule cell progenitors (GCPs) in the cerebellum, as was shown in Jdp2-promoter-Cre transgenic mice. Cerebellum of Jdp2-knockout (KO) mice contains lower number of Atoh-1 positive GCPs than WT. Primary cultures of GCPs from Jdp2-KO mice at postnatal day 5 were more resistant to apoptosis than GCPs from wild-type mice. In Jdp2-KO GCPs, the levels of both the glutamateâcystine exchanger Sc7a11 and glutathione were increased; by contrast, the activity of reactive oxygen species (ROS) was decreased; these changes confer resistance to ROS-mediated apoptosis. In the absence of Jdp2, a complex of the cyclin-dependent kinase inhibitor 1 (p21Cip1) and Nrf2 bound to antioxidant response elements of the Slc7a11 promoter and provide redox control to block ROS-mediated apoptosis. These findings suggest that an interplay between Jdp2, Nrf2, and p21Cip1 regulates the GCP apoptosis, which is one of critical events for normal development of the cerebellum.
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Obtaining differentiated cells with high physiological functions by an efficient, but simple and rapid differentiation method is crucial for modeling neuronal diseases in vitro using human pluripotent stem cells (hPSCs). Currently, methods involving the transient expression of one or a couple of transcription factors have been established as techniques for inducing neuronal differentiation in a rapid, single step. It has also been reported that microRNAs can function as reprogramming effectors for directly reprogramming human dermal fibroblasts to neurons. In this study, we tested the effect of adding neuronal microRNAs, miRNA-9/9*, and miR-124 (miR-9/9*-124), for the neuronal induction method of hPSCs using Tet-On-driven expression of the Neurogenin2 gene (Ngn2), a proneural factor. While it has been established that Ngn2 can facilitate differentiation from pluripotent stem cells into neurons with high purity due to its neurogenic effect, a long or indefinite time is required for neuronal maturation with Ngn2 misexpression alone. With the present method, the cells maintained a high neuronal differentiation rate while exhibiting increased gene expression of neuronal maturation markers, spontaneous calcium oscillation, and high electrical activity with network bursts as assessed by a multipoint electrode system. Moreover, when applying this method to iPSCs from Alzheimer's disease (AD) patients with presenilin-1 (PS1) or presenilin-2 (PS2) mutations, cellular phenotypes such as increased amount of extracellular secretion of amyloid ß42, abnormal oxygen consumption, and increased reactive oxygen species in the cells were observed in a shorter culture period than those previously reported. Therefore, it is strongly anticipated that the induction method combining Ngn2 and miR-9/9*-124 will enable more rapid and simple screening for various types of neuronal disease phenotypes and promote drug discovery.
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MicroRNAs/metabolismo , Doenças do Sistema Nervoso/genética , Neurogênese/fisiologia , Neurônios/metabolismo , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , Humanos , Neurônios/citologia , Fenótipo , TransfecçãoRESUMO
Glioblastoma is the most aggressive brain tumor characterized by diffuse infiltration into the normal brain parenchyma. Neural stem cells are known to possess the tumor-tropic migratory capacity and thus can be used as cellular vehicles for targeted delivery of therapeutic agents. In the present study, we evaluated the efficacy of herpes simplex virus thymidine kinase (HSV-TK) suicide gene therapy for glioblastoma using neural stem/progenitor cells (NS/PCs) derived from human induced pluripotent stem cells (hiPSCs). Although transduction of hiPSCs is preferable for a safe and stable supply in the clinical setting, high-level and/or constitutive HSV-TK expression was highly cytotoxic to hiPSCs. To overcome this problem, we used the tetracycline-inducible system to control the expression of HSV-TK. hiPSC-derived NS/PCs expressing HSV-TK were transplanted in an orthotopic xenograft mouse model of human glioblastoma. Glioblastoma cell growth in mice was dramatically inhibited following ganciclovir (GCV) administration. Survival of the mice was significantly prolonged with administration of GCV compared with control groups. Time-lapse imaging of organotypic brain slice cultures first demonstrated the directional migration of NS/PCs toward glioblastoma cells and the bystander killing effect upon GCV treatment. hiPSC-derived NS/PCs with HSV-TK/GCV suicide gene system may have considerable therapeutic potential for the treatment of glioblastoma. Color images are available online.
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Efeito Espectador , Terapia Genética , Glioblastoma/genética , Glioblastoma/terapia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Modelos Animais de Doenças , Feminino , Ganciclovir/administração & dosagem , Vetores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Simplexvirus/genética , Timidina Quinase/genética , Imagem com Lapso de Tempo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Chronic inflammation is accompanied by recurring cycles of tissue destruction and repair and is associated with an increased risk of cancer1-3. However, how such cycles affect the clonal composition of tissues, particularly in terms of cancer development, remains unknown. Here we show that in patients with ulcerative colitis, the inflamed intestine undergoes widespread remodelling by pervasive clones, many of which are positively selected by acquiring mutations that commonly involve the NFKBIZ, TRAF3IP2, ZC3H12A, PIGR and HNRNPF genes and are implicated in the downregulation of IL-17 and other pro-inflammatory signals. Mutational profiles vary substantially between colitis-associated cancer and non-dysplastic tissues in ulcerative colitis, which indicates that there are distinct mechanisms of positive selection in both tissues. In particular, mutations in NFKBIZ are highly prevalent in the epithelium of patients with ulcerative colitis but rarely found in both sporadic and colitis-associated cancer, indicating that NFKBIZ-mutant cells are selected against during colorectal carcinogenesis. In further support of this negative selection, we found that tumour formation was significantly attenuated in Nfkbiz-mutant mice and cell competition was compromised by disruption of NFKBIZ in human colorectal cancer cells. Our results highlight common and discrete mechanisms of clonal selection in inflammatory tissues, which reveal unexpected cancer vulnerabilities that could potentially be exploited for therapeutics in colorectal cancer.
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Proteínas Adaptadoras de Transdução de Sinal/genética , Colite Ulcerativa/genética , Taxa de Mutação , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular Tumoral , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Neoplasias Colorretais/genética , Humanos , Camundongos , Transdução de SinaisRESUMO
A lack of caudal-type homeobox transcription factor 2 (CDX2) protein expression has been proposed as a prognostic biomarker for colorectal cancer (CRC). However, the relationship between CDX2 levels and the survival of patients with stage II/III CRC along with the relationship between microRNAs (miRs) and CDX2 expression are unclear. Tissue samples were collected from patients with stage II/III CRC surgically treated at Kyoto University Hospital. CDX2 expression was semi-quantitatively evaluated by immunohistochemistry (IHC). The prognostic impacts of CDX2 expression on overall survival (OS) and relapse-free survival (RFS) were evaluated by multivariable statistical analysis. The expression of miRs regulating CDX2 expression and their prognostic impacts were analyzed using The Cancer Genome Atlas Program for CRC (TCGA-CRC). Eleven of 174 CRC tissues lacked CDX2 expression. The five-year OS and RFS rates of patients with CDX2-negative CRC were significantly lower than those of CDX2-positive patients. Multivariate analysis of clinicopathological features revealed that CDX2-negative status is an independent marker of poor prognosis in stage II/III CRC. miR-9-5p was shown to regulate CDX2 expression. TCGA-CRC analysis showed that high miR-9-5p expression was significantly associated with poor patient prognosis in stage II/III CRC. In conclusion, CDX2, the post-transcriptional target of microRNA-9-5p, is a useful prognostic biomarker in patients with stage II/III CRC.
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Cancer stem cell (CSC)-specific markers may be potential therapeutic targets. We previously identified that Dclk1, a tuft cell marker, marks tumor stem cells (TSCs) in mouse intestinal adenomas. Based on the analysis of mouse Dclk1+ tumor cells, we aimed to identify a CSC-specific cell surface marker in human colorectal cancers (hCRCs) and validate the therapeutic effect of targeting it. IL17RB was distinctively expressed by Dclk1+ mouse intestinal tumor cells. Using Il17rb-CreERT2-IRES-EGFP mice, we show that IL17RB marked intestinal TSCs in an IL13-dependent manner. Tuft cell-like cancer cells were detected in a subset of hCRCs. In these hCRCs, lineage-tracing experiments in CRISPR-Cas9-mediated IL17RB-CreERT2 knockin organoids and xenograft tumors revealed that IL17RB marks CSCs that expand independently of IL-13. We observed up-regulation of POU2F3, a master regulator of tuft cell differentiation, and autonomous tuft cell-like cancer cell differentiation in the hCRCs. Furthermore, long-term ablation of IL17RB-expressing CSCs strongly suppressed the tumor growth in vivo. These findings reveal insights into a CSC-specific marker IL17RB in a subset of hCRCs, and preclinically validate IL17RB+ CSCs as a cancer therapeutic target.
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Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Células-Tronco Neoplásicas/patologia , Receptores de Interleucina-17/metabolismo , Animais , Biomarcadores Tumorais/genética , Sistemas CRISPR-Cas/genética , Carcinogênese , Diferenciação Celular , Linhagem da Célula , Quinases Semelhantes a Duplacortina , Técnicas de Introdução de Genes , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Transgênicos , Fatores de Transcrição de Octâmero/metabolismo , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/metabolismo , Receptores de Interleucina-17/genética , Esferoides Celulares , Imagem com Lapso de Tempo , Células Tumorais Cultivadas , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Glioblastoma exhibits phenotypic and genetic heterogeneity, aggressive invasiveness, therapeutic resistance, and tumor recurrence, which can be explained by the existence of glioma stem cells (GSCs). In this study, we visualized the spatiotemporal dynamics of invasion of human GSCs in an orthotopic xenograft mouse model using time-lapse imaging of organotypic brain slice cultures and three-dimensional imaging of optically cleared whole brains. GSCs implanted in the striatum exhibited directional migration toward axon bundles, perivascular area, and the subventricular zone around the inferior horn of the lateral ventricle. GSCs migrated in a helical pattern around axon bundles in the striatum and invaded broadly in both the rostral and caudal directions. GSCs in the corpus callosum migrated more rapidly and unidirectionally toward the contralateral side with pseudopod extension. These characteristics of GSC invasion shared histological features observed in glioblastoma patients. Spatiotemporal visualization techniques can contribute to the elucidation of the mechanisms underlying GSC invasion that may lead to the development of effective therapy for glioblastoma.
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Neoplasias Encefálicas/patologia , Glioma/patologia , Células-Tronco Neoplásicas/patologia , Animais , Encéfalo/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Humanos , Imageamento Tridimensional , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Imagem com Lapso de TempoRESUMO
Nonhuman primates (NHPs) are considered to be the most valuable models for human transgenic (Tg) research into disease because human pathology is more closely recapitulated in NHPs than rodents. Previous studies have reported the generation of Tg NHPs that ubiquitously overexpress a transgene using various promoters, but it is not yet clear which promoter is most suitable for the generation of NHPs overexpressing a transgene ubiquitously and persistently in various tissues. To clarify this issue, we evaluated four putative ubiquitous promoters, cytomegalovirus (CMV) immediate-early enhancer and chicken beta-actin (CAG), elongation factor 1α (EF1α), ubiquitin C (UbC), and CMV, using an in vitro differentiation system of cynomolgus monkey embryonic stem cells (ESCs). While the EF1α promoter drove Tg expression more strongly than the other promoters in undifferentiated pluripotent ESCs, the CAG promoter was more effective in differentiated cells such as embryoid bodies and ESC-derived neurons. When the CAG and EF1α promoters were used to generate green fluorescent protein (GFP)-expressing Tg monkeys, the CAG promoter drove GFP expression in skin and hematopoietic tissues more strongly than in ΕF1α-GFP Tg monkeys. Notably, the EF1α promoter underwent more silencing in both ESCs and Tg monkeys. Thus, the CAG promoter appears to be the most suitable for ubiquitous and stable expression of transgenes in the differentiated tissues of Tg cynomolgus monkeys and appropriate for the establishment of human disease models.