RESUMO
Since various imaging modalities have been developed, cancer metastasis can be detected from an early stage. However, limitations still exist, especially in terms of spatial resolution. Tissue-clearing technology has emerged as a new imaging modality in cancer research, which has been developed and utilized for a long time mainly in neuroscience field. This method enables us to detect cancer metastatic foci with single-cell resolution at whole mouse body/organ level. On top of that, 3D images of cancer metastasis of whole mouse organs make it easy to understand their characteristics. Recently, further applications of tissue clearing methods were reported in combination with reporter systems, labeling, and machine learning. In this review, we would like to provide an overview of this technique and current applications in cancer research and discuss their potentials and limitations.
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Progressão da Doença , Imageamento Tridimensional , Metástase Neoplásica , Neoplasias , Animais , Camundongos , Humanos , Neoplasias/patologia , Neoplasias/diagnóstico por imagem , Imageamento Tridimensional/métodos , Aprendizado de MáquinaRESUMO
Transforming growth factor ß (TGF-ß) is a pleiotropic cytokine that is widely distributed throughout the body. Its receptor proteins, TGF-ß type I and type II receptors, are also ubiquitously expressed. Therefore, the regulation of various signaling outputs in a context-dependent manner is a critical issue in this field. Smad proteins were originally identified as signal-activated transcription factors similar to signal transducer and activator of transcription proteins. Smads are activated by serine phosphorylation mediated by intrinsic receptor dual specificity kinases of the TGF-ß family, indicating that Smads are receptor-restricted effector molecules downstream of ligands of the TGF-ß family. Smad proteins have other functions in addition to transcriptional regulation, including post-transcriptional regulation of micro-RNA processing, pre-mRNA splicing, and m6A methylation. Recent technical advances have identified a novel landscape of Smad-dependent signal transduction, including regulation of mitochondrial function without involving regulation of gene expression. Therefore, Smad proteins are receptor-activated transcription factors and also act as intracellular signaling modulators with multiple modes of function. In this review, we discuss the role of Smad proteins as receptor-activated transcription factors and beyond. We also describe the functional differences between Smad2 and Smad3, two receptor-activated Smad proteins downstream of TGF-ß, activin, myostatin, growth and differentiation factor (GDF) 11, and Nodal.
Assuntos
Transdução de Sinais , Proteína Smad2 , Proteína Smad3 , Fator de Crescimento Transformador beta , Animais , Humanos , Proteína Smad2/metabolismo , Proteína Smad2/genética , Proteína Smad3/metabolismo , Proteína Smad3/genética , Fator de Crescimento Transformador beta/metabolismo , Ligação Proteica , Cromatina/genética , Cromatina/metabolismo , Transcrição GênicaRESUMO
The tumor microenvironment (TME) consists of cancer cells surrounded by stromal components including tumor vessels. Transforming growth factor-ß (TGF-ß) promotes tumor progression by inducing epithelial-mesenchymal transition (EMT) in cancer cells and stimulating tumor angiogenesis in the tumor stroma. We previously developed an Fc chimeric TGF-ß receptor containing both TGF-ß type I (TßRI) and type II (TßRII) receptors (TßRI-TßRII-Fc), which trapped all TGF-ß isoforms and suppressed tumor growth. However, the precise mechanisms underlying this action have not yet been elucidated. In the present study, we showed that the recombinant TßRI-TßRII-Fc protein effectively suppressed in vitro EMT of oral cancer cells and in vivo tumor growth in a human oral cancer cell xenograft mouse model. Tumor cell proliferation and angiogenesis were suppressed in tumors treated with TßRI-TßRII-Fc. Molecular profiling of human cancer cells and mouse stroma revealed that K-Ras signaling and angiogenesis were suppressed. Administration of TßRI-TßRII-Fc protein decreased the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), interleukin-1ß (IL-1ß) and epiregulin (EREG) in the TME of oral cancer tumor xenografts. HB-EGF increased proliferation of human oral cancer cells and mouse endothelial cells by activating ERK1/2 phosphorylation. HB-EGF also promoted oral cancer cell-derived tumor formation by enhancing cancer cell proliferation and tumor angiogenesis. In addition, increased expressions of IL-1ß and EREG in oral cancer cells significantly enhanced tumor formation. These results suggest that TGF-ß signaling in the TME controls cancer cell proliferation and angiogenesis by activating HB-EGF/IL-1ß/EREG pathways and that TßRI-TßRII-Fc protein is a promising tool for targeting the TME networks.
Assuntos
Neoplasias Bucais , Proteínas Serina-Treonina Quinases , Humanos , Camundongos , Animais , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Células Endoteliais/metabolismo , Microambiente Tumoral , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta1 , Neoplasias Bucais/genética , Fatores de Crescimento TransformadoresRESUMO
Transforming growth factor ß (TGF-ß) increases epithelial cancer cell migration and metastasis by inducing epithelial-mesenchymal transition (EMT). TGF-ß also inhibits cell proliferation by inducing G1 phase cell-cycle arrest. However, the correlation between these tumor-promoting and -suppressing effects remains unclear. Here, we show that TGF-ß confers higher motility and metastatic ability to oral cancer cells in G1 phase. Mechanistically, keratin-associated protein 2-3 (KRTAP2-3) is a regulator of these dual effects of TGF-ß, and its expression is correlated with tumor progression in patients with head and neck cancer and migratory and metastatic potentials of oral cancer cells. Furthermore, single-cell RNA sequencing reveals that TGF-ß generates two populations of mesenchymal cancer cells with differential cell-cycle status through two distinctive EMT pathways mediated by Slug/HMGA2 and KRTAP2-3. Thus, TGF-ß-induced KRTAP2-3 orchestrates cancer cell proliferation and migration by inducing EMT, suggesting motile cancer cells arrested in G1 phase as a target to suppress metastasis.
Assuntos
Neoplasias Bucais , Fator de Crescimento Transformador beta , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal/genética , Pontos de Checagem da Fase G1 do Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Queratinas/metabolismo , Neoplasias Bucais/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
The blood and lymphatic vasculature networks are not yet fully understood even in mouse because of the inherent limitations of imaging systems and quantification methods. This study aims to evaluate the usefulness of the tissue-clearing technology for visualizing blood and lymphatic vessels in adult mouse. Clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) enables us to capture the high-resolution 3D images of organ- or area-specific vascular structures. To evaluate these 3D structural images, signals are first classified from the original captured images by machine learning at pixel base. Then, these classified target signals are subjected to topological data analysis and non-homogeneous Poisson process model to extract geometric features. Consequently, the structural difference of vasculatures is successfully evaluated in mouse disease models. In conclusion, this study demonstrates the utility of CUBIC for analysis of vascular structures and presents its feasibility as an analysis modality in combination with 3D images and mathematical frameworks.
Assuntos
Análise de Dados , Vasos Linfáticos , Animais , Encéfalo/diagnóstico por imagem , Imageamento Tridimensional/métodos , Vasos Linfáticos/diagnóstico por imagem , Camundongos , TecnologiaRESUMO
Bone morphogenetic proteins (BMPs), members of the transforming growth factor-ß (TGF-ß) family, are multifunctional cytokines. BMPs have a broad range of functions, and abnormalities in BMP signaling pathways are involved in cancer progression. BMPs activate the proliferation of certain cancer cells. Malignant phenotypes of cancer cells, such as increased motility, invasiveness, and stemness, are enhanced by BMPs. Simultaneously, BMPs act on various cellular components and regulate angiogenesis in the tumor microenvironment. Thus, BMPs function as pro-tumorigenic factors in various types of cancer. However, similar to TGF-ß, which shows both positive and negative effects on tumorigenesis, BMPs also act as tumor suppressors in other types of cancers. In this article, we review important findings published in the recent decade and summarize the pro-oncogenic functions of BMPs and their underlying mechanisms. The current status of BMP-targeted therapies for cancers is also discussed.
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We developed an in vivo serial passaging model for renal cancer with orthotopic renal subcapsular inoculation. We detail the procedures for renal subcapsular inoculation of cancer cells in mice, followed by in vivo and exvivo bioluminescence imaging, tumor-bearing kidney dissociation, and in vivo passaging. This protocol is capable of reproducing the coevolution between cancer cells and the primary tumor microenvironment. It enables us to unveil the systemic dynamics of metastasis and develop a therapeutic strategy for metastatic renal cancer. For complete details on the use and execution of this protocol, please refer to Nishida et al. (2020).
Assuntos
Neoplasias Renais , Transplantes , Animais , Diagnóstico por Imagem , Feminino , Humanos , Rim/patologia , Neoplasias Renais/patologia , Masculino , Camundongos , Transplantes/patologia , Microambiente TumoralRESUMO
Transforming growth factor ß (TGFß) induces epithelial-mesenchymal transition (EMT), which correlates with stemness and invasiveness. Mesenchymal-epithelial transition (MET) is induced by TGFß withdrawal and correlates with metastatic colonization. Whether TGFß promotes stemness and invasiveness simultaneously via EMT remains unclear. We established a breast cancer cell model expressing red fluorescent protein (RFP) under the E-cadherin promoter. In 2D cultures, TGFß induced EMT, generating RFPlow cells with a mesenchymal transcriptome, and regained RFP, with an epithelial transcriptome, after MET induced by TGFß withdrawal. RFPlow cells generated robust mammospheres, with epithelio-mesenchymal cell surface features. Mammospheres that were forced to adhere generated migratory cells, devoid of RFP, a phenotype which was inhibited by a TGFß receptor kinase inhibitor. Further stimulation of RFPlow mammospheres with TGFß suppressed the generation of motile cells, but enhanced mammosphere growth. Accordingly, mammary fat-pad-transplanted mammospheres, in the absence of exogenous TGFß treatment, established lung metastases with evident MET (RFPhigh cells). In contrast, TGFß-treated mammospheres revealed high tumour-initiating capacity, but limited metastatic potential. Thus, the biological context of partial EMT and MET allows TGFß to differentiate between pro-stemness and pro-invasive phenotypes.
Assuntos
Transição Epitelial-Mesenquimal , Neoplasias Pulmonares , Linhagem Celular Tumoral , Humanos , Fenótipo , Receptores de Fatores de Crescimento Transformadores beta , Fator de Crescimento Transformador beta/metabolismoRESUMO
I would like to congratulate the Journal of Biochemistry (JB) on its 100th anniversary. I served as the Editor-in-Chief for four years starting in 2010 and succeeded Dr Naoyuki Taniguchi.
RESUMO
The activation of RIG-I-like receptor (RLR) signaling in cancer cells is widely recognized as a critical cancer therapy method. The expected mechanism of RLR ligand-mediated cancer therapy involves the promotion of cancer cell death and strong induction of interferon (IFN)-ß that affects the tumor microenvironment. We have recently shown that activation of RLR signaling in triple-negative breast cancer cells (TNBC) attenuates transforming growth factor-ß (TGF-ß) signaling, which partly contributes to the promotion of cancer cell pyroptosis. However, the consequences of suppression of TGF-ß signaling by RLR ligands with respect to IFN-ß-mediated tumor suppression are not well characterized. This study showed that transfection of a typical RLR ligand polyI:C in cancer cells produces significant levels of IFN-ß, which inhibits the growth of the surrounding cancer cells. In addition, IFN-ß-induced cell cycle arrest in surrounding cancer cells was inhibited by the expression of constitutively active Smad3. Constitutively active Smad3 suppresses IFN-ß expression through the alleviation of IFN regulatory factor 3 binding to the canonical target genes, as suggested by ChIP sequencing analysis. Based on these findings, a new facet of the protumorigenic function of TGF-ß that suppresses IFN-ß expression is suggested when RLR-mediated cancer treatment is used in TNBC.
Assuntos
Interferon beta/metabolismo , Poli I-C/farmacologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Fator Regulador 3 de Interferon/metabolismo , Poli I-C/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína Smad3/genética , Proteína Smad3/metabolismo , Transfecção , Fator de Crescimento Transformador beta/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
Smad proteins transduce signals downstream of transforming growth factor-ß (TGF-ß) and are one of the factors that regulate the expression of genes related to diseases affecting the skin. In the present study, we identified MAB21L4, also known as male abnormal 21 like 4 or C2orf54, as the most up-regulated targets of TGF-ß and Smad3 in differentiated human progenitor epidermal keratinocytes using chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq). We found that TGF-ß induced expression of the barrier protein involucrin (encoded by the IVL gene). Transcriptional activity of the IVL promoter induced by TGF-ß was inhibited by MAB21L4 siRNAs. Further analysis revealed that MAB21L4 siRNAs also down-regulated the expression of several target genes of TGF-ß. MAB21L4 protein was located mainly in the cytosol, where it was physically bound to Smad3 and a transcriptional corepressor c-Ski. siRNAs for MAB21L4 did not inhibit the binding of Smad3 to their target genomic regions but down-regulated the acetylation of histone H3 lys 27 (H3K27ac), an active histone mark, near the Smad3 binding regions. These findings suggest that TGF-ß-induced MAB21L4 up-regulates the gene expression induced by TGF-ß, possibly through the inhibition of c-Ski via physical interaction in the cytosol.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta , Humanos , Queratinócitos/metabolismo , Masculino , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Glioma-initiating cells (GICs), a major source of glioblastoma recurrence, are characterized by the expression of neural stem cell markers and the ability to grow by forming nonadherent spheres under serum-free conditions. Bone morphogenetic proteins (BMPs), members of the transforming growth factor-ß family, induce differentiation of GICs and suppress their tumorigenicity. However, the mechanisms underlying the BMP-induced loss of GIC stemness have not been fully elucidated. Here, we show that paired related homeobox 1 (PRRX1) induced by BMPs decreases the CD133-positive GIC population and inhibits tumorigenic activity of GICs in vivo. Of the two splice isoforms of PRRX1, the longer isoform, pmx-1b, but not the shorter isoform, pmx-1a, induces GIC differentiation. Upon BMP stimulation, pmx-1b interacts with the DNA methyltransferase DNMT3A and induces promoter methylation of the PROM1 gene encoding CD133. Silencing DNMT3A maintains PROM1 expression and increases the CD133-positive GIC population. Thus, pmx-1b promotes loss of stem cell-like properties of GICs through region-specific epigenetic regulation of CD133 expression by recruiting DNMT3A, which is associated with decreased tumorigenicity of GICs.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , DNA Metiltransferase 3A , Epigênese Genética , Glioblastoma/genética , Glioblastoma/metabolismo , Glioma/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Neoplásicas/metabolismoRESUMO
DNA hypermethylation is frequently observed in clear cell renal cell carcinoma (ccRCC) and correlates with poor clinical outcomes. However, the detailed function of DNA hypermethylation in ccRCC has not been fully uncovered. Here, we show the role of DNA methylation in ccRCC progression through the identification of a target(s) of DNA methyltransferases (DNMT). Our preclinical model of ccRCC using the serial orthotopic inoculation model showed the upregulation of DNMT3B in advanced ccRCC. Pretreatment of advanced ccRCC cells with 5-aza-deoxycytidine, a DNMT inhibitor, attenuated the formation of primary tumors through the induction of apoptosis. DNA methylated sites were analyzed genome-wide using methylation array in reference to RNA-sequencing data. The gene encoding ubiquinol cytochrome c reductase hinge protein (UQCRH), one of the components of mitochondrial complex III, was extracted as a methylation target in advanced ccRCC. Immunohistochemical analysis revealed that the expression of UQCRH in human ccRCC tissues was lower than normal adjacent tissues. Silencing of UQCRH attenuated the cytochrome c release in response to apoptotic stimuli and resulted in enhancement of primary tumor formation in vivo, implying the tumor-suppressive role of UQCRH. Moreover, 5-aza-deoxycytidine enhanced the therapeutic efficiency of mammalian target of rapamycin inhibitor everolimus in vivo. These findings suggested that the DNMT3B-induced methylation of UQCRH may contribute to renal cancer progression and implicated clinical significance of DNMT inhibitor as a therapeutic option for ccRCC.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Apoptose/genética , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Metilação de DNA/genética , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Neoplasias Renais/patologiaRESUMO
Follistatin-like 3 (FSTL3) is an endogenous antagonist against transforming growth factor-ß family ligands. Monovalent FSTL3-Fc fusion protein (mono-FSTL3-Fc) generated with knobs-into-holes technology overcomes limitations of current anti-myostatin therapies. We have developed a facile protocol for affinity purification of the Fc-fused protein from the supernatant of HEK293T cells stably expressing the protein. This protocol is advantageous by only requiring readily accessible equipment. We further outline the steps for validation of mono-FSTL3-Fc increasing systemic muscle mass in mice after intraperitoneal administration. For complete details on the use and execution of this protocol, please refer to Ozawa et al. (2021).
Assuntos
Proteínas Relacionadas à Folistatina , Folistatina , Animais , Folistatina/metabolismo , Proteínas Relacionadas à Folistatina/genética , Células HEK293 , Humanos , Camundongos , Músculos/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
We previously reported that bone morphogenetic protein (BMP) signaling promotes tumorigenesis in gynecologic cancer cells. BMP2 enhances proliferation of ovarian and endometrial cancer cells via c-KIT induction, and triggers epithelial-mesenchymal transition (EMT) by SNAIL and/or SLUG induction, leading to increased cell migration. However, the downstream effectors of BMP signaling in gynecological cancer cells have not been clearly elucidated. In this study, we performed RNA-sequencing of Ishikawa endometrial and SKOV3 ovarian cancer cells after BMP2 stimulation, and identified TNFRSF12A, encoding fibroblast growth factor-inducible 14 (FN14) as a common BMP2-induced gene. FN14 knockdown suppressed BMP2-induced cell proliferation and migration, confirmed by MTS and scratch assays, respectively. In addition, FN14 silencing augmented chemosensitivity of SKOV3 cells. As a downstream effector of BMP signaling, FN14 modulated both c-KIT and SNAIL expression, which are important for growth and migration of ovarian and endometrial cancer cells. These results support the notion that the tumor promoting effects of BMP signaling in gynecological cancers are partially attributed to FN14 induction.
Assuntos
Neoplasias do Endométrio , Neoplasias Ovarianas , Transdução de Sinais , Proteína Morfogenética Óssea 2/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Transição Epitelial-Mesenquimal , Feminino , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Receptor de TWEAKRESUMO
The effects of bone morphogenetic proteins (BMPs), members of the transforming growth factor-ß (TGF-ß) family, in endometrial cancer (EC) have yet to be determined. In this study, we analyzed the TCGA and MSK-IMPACT datasets and investigated the effects of BMP2 and of TWSG1, a BMP antagonist, on Ishikawa EC cells. Frequent ACVR1 mutations and high mRNA expressions of BMP ligands and receptors were observed in EC patients of the TCGA and MSK-IMPACT datasets. Ishikawa cells secreted higher amounts of BMP2 compared with ovarian cancer cell lines. Exogenous BMP2 stimulation enhanced EC cell sphere formation via c-KIT induction. BMP2 also induced EMT of EC cells, and promoted migration by induction of SLUG. The BMP receptor kinase inhibitor LDN193189 augmented the growth inhibitory effects of carboplatin. Analyses of mRNAs of several BMP antagonists revealed that TWSG1 mRNA was abundantly expressed in Ishikawa cells. TWSG1 suppressed BMP7-induced, but not BMP2-induced, EC cell sphere formation and migration. Our results suggest that BMP signaling promotes EC tumorigenesis, and that TWSG1 antagonizes BMP7 in EC. BMP signaling inhibitors, in combination with chemotherapy, might be useful in the treatment of EC patients.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 7/metabolismo , Carcinógenos/metabolismo , Neoplasias do Endométrio/patologia , Regulação Neoplásica da Expressão Gênica , Apoptose , Biomarcadores Tumorais/genética , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 7/genética , Proliferação de Células , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Feminino , Humanos , Prognóstico , Transdução de Sinais , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
Intratumoral heterogeneity, including in clear cell renal cell carcinoma, is a potential cause of drug resistance and metastatic cancer progression. We specified the heterogeneous population marked by endoglin (also known as CD105) in a preclinical model of clear cell renal cell carcinoma progression. Highly malignant derivatives of human clear cell renal cell carcinoma OS-RC-2 cells were established as OS5Ks by serial orthotopic inoculation in our previous study. Expression of both ENG (encoding endoglin) mRNA and protein were heterogeneously upregulated in OS5Ks, and the endoglin-positive (ENG+ ) population exhibited growth dependency on endoglin in anchorage-independent cultures. Despite the function of endoglin as a type III receptor, transforming growth factor ß and bone morphogenetic protein-9 signaling were unlikely to contribute to the proliferative phenotype. Although endoglin has been proposed as a marker for renal cancer-initiating cells, the OS5K-3 ENG+ population did not enrich other reported cancer-initiating cell markers or differentiate into the ENG- population. Mouse tumor inoculation models revealed that the tumor-forming capabilities of OS5K-3 ENG+ and ENG- cells in vivo were highly dependent on the microenvironment, with the renal microenvironment most preferable to ENG+ cells. In conclusion, the renal microenvironment, rather than the hypothesized ENG+ cell-centered hierarchy, maintains cellular heterogeneity in clear cell renal cell carcinoma. Therefore, the effect of the microenvironment should be considered when evaluating the proliferative capability of renal cancer cells in the experimental settings.
Assuntos
Carcinoma de Células Renais/patologia , Endoglina/genética , Endoglina/metabolismo , Neoplasias Renais/patologia , Regulação para Cima , Animais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Masculino , Camundongos , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Microambiente TumoralRESUMO
Tissue-clearing technology is an emerging imaging technique currently utilized not only in neuroscience research but also in cancer research. In our previous reports, tissue-clearing methods were used for the detection of metastatic tumors. Here, we showed that the cell cycles of primary and metastatic tumors were visualized by tissue-clearing methods using a reporter system. First, we established cancer cell lines stably expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) reporter with widely used cancer cell lines A549 and 4T1. Fluorescence patterns of the Fucci reporter were investigated in various tumor inoculation models in mice. Interestingly, fluorescence patterns of the Fucci reporter of tumor colonies were different between various organs, and even among colonies in the same organs. The effects of antitumor drugs were also evaluated using these Fucci reporter cells. Of the three antitumor drugs studied, 5-fluorouracil treatment on 4T1-Fucci cells resulted in characteristic fluorescent patterns by the induction of G2 /M arrest both in vitro and in vivo. Thus, the combination of a tissue-clearing method with the Fucci reporter is useful for analyzing the mechanisms of cancer metastasis and drug resistance.
Assuntos
Adenocarcinoma de Pulmão/patologia , Neoplasias da Mama/patologia , Ciclo Celular , Medições Luminescentes/métodos , Neoplasias Pulmonares/patologia , Células A549 , Animais , Antimetabólitos Antineoplásicos/administração & dosagem , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Feminino , Fluoruracila/administração & dosagem , Genes Reporter , Vetores Genéticos/genética , Humanos , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia de Fluorescência/métodos , Transfecção , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína Vermelha FluorescenteRESUMO
Targeting the signaling pathway of growth differentiation factor 8 (GDF8), also known as myostatin, has been regarded as a promising strategy to increase muscle mass in the elderly and in patients. Accumulating evidence in animal models and clinical trials has indicated that a rational approach is to inhibit a limited number of transforming growth factor ß (TGF-ß) family ligands, including GDF8 and activin A, without affecting other members. Here, we focused on one of the endogenous antagonists against TGF-ß family ligands, follistatin-like 3 (FSTL3), which mainly binds and neutralizes activins, GDF8, and GDF11. Although bivalent human FSTL3 Fc-fusion protein was rapidly cleared from mouse circulation similar to follistatin (FST)-Fc, monovalent FSTL3-Fc (mono-FSTL3-Fc) generated with the knobs-into-holes technology exhibited longer serum half-life. Systemic administration of mono-FSTL3-Fc in mice induced muscle fiber hypertrophy and increased muscle mass in vivo. Our results indicate that the monovalent FSTL3-based therapy overcomes the difficulties of current anti-GDF8 therapies.