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1.
Tumour Biol ; 37(8): 11267-78, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26951511

RESUMO

Dendritic cell (DC) vaccination targeting cancer stem cells is an effective way to suppress tumor progression and reduce the metastasis and recurrence. In the present study, we explored the suitability of side population (SP) cells as source of antigens for DC vaccination against hepatocellular carcinoma (HCC) in a mouse model. In this study, we identified the "stem-like" characteristics of SP cells in the MHCC97 and Hepa 1-6 HCC cell lines. We found that SP cells express high levels of tumor-associated antigens and MHC class I molecules. Although loading with cell lysates did not change the characteristics of DCs, SP cell lysate-pulsed DCs induced antigen-specific T cell responses, including T cell proliferation and increased IFN-γ production by stimulated CD8(+) T cells. We investigated the cytotoxicity of T cells stimulated by SP cell lysate-pulsed DCs in nude mice co-injected with MHCC97 cells. To mimic the in vivo environment, we also confirmed the result in mouse HCC cell line Hepa 1-6 induced tumor-bearing C57/BL6 immune competent mice, and we demonstrated that vaccination with DCs loaded with Hepa 1-6 SP cell lysates could induce a T cell response in vivo and suppress the tumor growth. Our results may have applications for anti-HCC immunotherapy by targeting the cancer stem cells and may provide new insight for cancer vaccines.


Assuntos
Antígenos de Neoplasias/imunologia , Carcinoma Hepatocelular/imunologia , Células Dendríticas/imunologia , Neoplasias Hepáticas/imunologia , Células da Side Population/imunologia , Linfócitos T/imunologia , Animais , Western Blotting , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imunoterapia/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Células-Tronco Neoplásicas/imunologia , Reação em Cadeia da Polimerase , Vacinação , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Stem Cell Res ; 11(1): 625-33, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23651585

RESUMO

This report brings the first experimental evidence for the presence of long-term (LT) repopulating hematopoietic stem cells (HSCs) and Side Population (SP) cells within human steady state peripheral blood CD34(+) cells. Ex vivo culture, which reveals the LT-HSC, also increases short-term (ST) HSC engraftment capacity and SP cell number (as well as the SP subpopulations defined on the basis of CD38, CD90 and CD133 expression) which are very low in freshly isolated cells. Thus, ex vivo incubation either allows the expansion of the small fraction of HSCs or reveals "Scid Repopulating Cells - SRC" that are present in the initial CD34(+) cell population but unable to engraft. In addition, among these CD34(+) cells, we confirm the presence of committed progenitors at frequencies similar to those found in cord blood CD34(+) cells. These cells, obtained from leukoreduction filters (LRFs) and rejected in the course of the preparation of red blood cell concentrates, are an abundant and reliable material for obtaining committed progenitors, short- and long-term HSCs of therapeutic interest, especially after the ex vivo expansion phase. Our results open a perspective to set up new therapeutic protocols using expanded LRFs-recovered CD34(+) cells as a source of HSCs for autologous or allogeneic transplantation.


Assuntos
Células-Tronco Hematopoéticas/citologia , Células da Side Population/citologia , Animais , Separação Celular , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células da Side Population/imunologia , Células da Side Population/metabolismo
3.
Stem Cells Dev ; 22(16): 2221-39, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23540661

RESUMO

Cancer stem cells (CSCs) constitute a subpopulation of cancer cells that have the potential for self-renewal, multipotent differentiation, and tumorigenicity. Studies on CSC biology and CSC-targeted therapies depend on CSC isolation and/or enrichment methodologies. Scientists have conducted extensive research in this field since John Dick's group successfully isolated CSCs based on the expression of the CD34 and CD38 surface markers. Progress in CSC research has been greatly facilitated by the enrichment and isolation of these cells. In this review, we summarize the current strategies used in our and other laboratories for CSC isolation and enrichment, including methods based on stem cell surface markers, intracellular enzyme activity, the concentration of reactive oxygen species, the mitochondrial membrane potential, promoter-driven fluorescent protein expression, autofluorescence, suspension/adherent culture, cell division, the identification of side population cells, resistance to cytotoxic compounds or hypoxia, invasiveness/adhesion, immunoselection, and physical property. Although many challenges remain to be overcome, it is reasonable to believe that more reliable, efficient, and convenient methods will be developed in the near future.


Assuntos
Separação Celular/métodos , Células-Tronco Neoplásicas/citologia , Células da Side Population/citologia , ADP-Ribosil Ciclase 1/imunologia , ADP-Ribosil Ciclase 1/metabolismo , Antígenos CD34/imunologia , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Adesão Celular , Técnicas de Cultura de Células , Diferenciação Celular , Centrifugação com Gradiente de Concentração , Humanos , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Potencial da Membrana Mitocondrial , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células da Side Population/imunologia , Células da Side Population/metabolismo , Transdução de Sinais
4.
Leukemia ; 26(9): 2124-34, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22430632

RESUMO

Cancer stem cells have been proposed to be responsible for tumorigenesis and recurrence in various neoplastic diseases, including multiple myeloma (MM). We have previously reported that MM cells specifically express HLA class I at high levels and that single-chain Fv diabody against this molecule markedly induces MM cell death. Here we investigated the effect of a new diabody (C3B3) on cancer stem cell-like side population (SP) cells. SP fraction of MM cells highly expressed ABCG2 and exhibited resistance to chemotherapeutic agents; however, C3B3 induced cytotoxicity in both SP cells and main population (MP) cells to a similar extent. Moreover, C3B3 suppressed colony formation and tumorigenesis of SP cells in vitro and in vivo. Crosslinking of HLA class I by C3B3 mediated disruption of lipid rafts and actin aggregation, which led to inhibition of gene expression of ß-catenin and pluripotency-associated transcription factors such as Sox2, Oct3/4 and Nanog. Conversely, knockdown of Sox2 and Oct3/4 mRNA reduced the proportion of SP cells, suggesting that these factors are essential in maintenance of SP fraction in MM cells. Thus, our findings reveal that immunotherapeutic approach by engineered antibodies can overcome drug resistance, and provide a new basis for development of cancer stem cell-targeted therapy.


Assuntos
Antígenos HLA/imunologia , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células da Side Population/metabolismo , Anticorpos de Cadeia Única/uso terapêutico , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/uso terapêutico , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos SCID , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/imunologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células da Side Population/imunologia , Células da Side Population/patologia , Anticorpos de Cadeia Única/imunologia , beta Catenina/metabolismo
5.
Methods Mol Biol ; 750: 47-59, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21618082

RESUMO

Hematopoietic stem cells (HSCs) are defined by the capabilities of multi-lineage differentiation and long-term self-renewal. Both these characteristics contribute to maintain the homeostasis of the system and allow the restoration of hematopoiesis after insults, such as infections or therapeutic ablation. Reconstitution after lethal irradiation strictly depends on a third, fundamental property of HSCs: the capability to migrate under the influence of specific chemokines. Directed by a chemotactic compass, after transplant HSCs find their way to the bone marrow, where they eventually home and engraft. HSCs represent a rare population that primarily resides in the bone marrow with an estimated frequency of 0.01% of total nucleated cells. Separating HSCs from differentiated cells that reside in the bone marrow has been the focus of intense investigation for years. In this chapter, we will describe in detail the strategy routinely used by our laboratory to purify murine HSCs, by exploiting their antigenic phenotype (KSL), combined with the physiological capability to efficiently efflux the vital dye Hoechst 33342, generating the so-called Side Population, or SP.


Assuntos
Benzimidazóis/metabolismo , Separação Celular/métodos , Citometria de Fluxo/métodos , Células-Tronco Hematopoéticas/citologia , Células da Side Population/citologia , Animais , Antígenos/imunologia , Antígenos/metabolismo , Medula Óssea/fisiologia , Diferenciação Celular , Linhagem da Célula/imunologia , Quimiotaxia , Corantes Fluorescentes/metabolismo , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/imunologia , Camundongos , Fenótipo , Células da Side Population/imunologia
6.
Blood ; 117(9): 2640-8, 2011 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-21220748

RESUMO

Dendritic cells (DCs) are known to regulate immune responses by inducing both central and peripheral tolerance. DCs play a vital role in negative selection of developing thymocytes by deleting T cells with high-affinity for self-peptide-major histocompatibility complexes. In the periphery, DCs mediate peripheral tolerance by promoting regulatory T-cell development, induction of T-cell unresponsiveness, and deletion of activated T cells. We studied whether allogeneic DCs, obtained from bone marrow cultured with either Flt3L (FLDCs) or granulocyte-macrophage colony-stimulating factor (GMDCs), could induce allospecific central and peripheral tolerance after IV injection; B cells were used as a control. The results showed that only FLDCs reached the thymus after injection and that these cells induced both central and peripheral tolerance to donor major histocompatibility complexes. For central tolerance, injection of FLDCs induced antigen-specific clonal deletion of both CD8 and CD4 single-positive thymocytes. For peripheral tolerance, injection of FLDCs induced donor-specific T-cell unresponsiveness and prolonged survival of donor-derived skin grafts. Tolerance induction by adoptive transfer of FLDCs could be a useful approach for promoting graft acceptance after organ transplantation.


Assuntos
Células Dendríticas/citologia , Células Dendríticas/imunologia , Sobrevivência de Enxerto/imunologia , Tolerância Imunológica/imunologia , Transplante de Pele/imunologia , Transferência Adotiva , Animais , Movimento Celular , Proliferação de Células , Células Clonais , Epitopos/imunologia , Injeções , Proteínas de Membrana/metabolismo , Camundongos , Células da Side Population/citologia , Células da Side Population/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Timo/citologia , Fatores de Tempo , Transplante Homólogo
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