RESUMEN
Development of new therapies for myeloma has been hindered by the lack of suitable preclinical animal models of the disease in which widespread tumor foci in the skeleton can be detected reliably. Traditional means of detecting skeletal tumor infiltration such as histopathology are cumbersome and labor-intensive and do not allow temporal monitoring of tumor progression or regression in response to therapy. To resolve this problem, we modified the Radl 5TGM1 model of myeloma bone disease such that fluorescent myeloma tumors can be optically imaged in situ. Here, we show that murine myeloma 5TGM1 tumor cells, engineered to express enhanced green fluorescent protein (eGFP; 5TGM1-eGFP cells), can be imaged in a temporal fashion using a fluorescence illuminator and a charge-coupled device camera in skeletons of live C57BL/KaLwRij mice. High-resolution, whole-body images of tumor-bearing mice revealed that myeloma cells homed almost exclusively to the skeleton, with multiple focal tumor foci in the axial skeleton, consistent with myeloma tumor distribution in humans. Finally, the tested antitumor treatment effect of Velcade (bortezomib), a proteasome inhibitor used clinically in myeloma, was readily detected by GFP imaging, suggesting the power of the technique in combination with the Radl 5TGM1-eGFP model for rapid preclinical assessment and sensitive monitoring of novel and potential therapeutics. Whole-body GFP imaging is practical, convenient, inexpensive, and rapid, and these advantages should enable a high throughput when evaluating in vivo efficacy of new potential antimyeloma therapeutics and assessing response to treatment.
Asunto(s)
Mieloma Múltiple/diagnóstico , Animales , Fluorescencia , Proteínas Fluorescentes Verdes/genética , RatonesRESUMEN
Parathyroid hormone-related peptide (PTHrP) is a major factor involved in tumor-induced osteolysis caused by breast cancers that have metastasized to bone. However, the molecular mechanisms that mediate PTHrP production by breast cancer cells are not entirely clear. We hypothesized that Gli2, a downstream transcriptional effector of the Hedgehog (Hh) signaling pathway, regulates PTHrP expression in metastatic breast cancer because the Hh pathway regulates physiologic PTHrP expression in the developing growth plate. Here, we show that Gli2 is expressed in several human cancer cell lines that cause osteolytic lesions in vivo and produce PTHrP (MDA-MB-231, RWGT2, and PC-3) but is not expressed in nonosteolytic cancer cell lines that do not secrete PTHrP (MCF-7, ZR-75, and T47D). Transient expression of Gli2 in MDA-MB-231 and MCF-7 breast cancer cells increased PTHrP promoter-luciferase activity dose dependently. Stable expression of Gli2 in MDA-MB-231 cells resulted in an increase in PTHrP protein in the conditioned medium. Alternatively, MDA-MB-231 cells stably transfected with Gli2-EnR, a repressor of Gli2 activity, exhibited a 72% to 93% decrease in PTHrP mRNA by quantitative real-time PCR when compared with control cells. To examine the effects of Gli2 on breast cancer-mediated osteolysis in vivo, athymic nude mice were inoculated with MDA-MB-231 cells stably expressing Gli2 or the empty vector. Following tumor cell inoculation via the left cardiac ventricle, Gli2-expressing tumors caused significantly more osteolysis. Together, these data suggest that PTHrP expression and osteolysis in vivo in human breast cancer cells is driven at least in part by Gli2.
Asunto(s)
Neoplasias de la Mama/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas Nucleares/metabolismo , Osteólisis/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/biosíntesis , Animales , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Medios de Cultivo Condicionados , Humanos , Factores de Transcripción de Tipo Kruppel/antagonistas & inhibidores , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Proteínas Nucleares/antagonistas & inhibidores , Osteólisis/patología , Proteína Relacionada con la Hormona Paratiroidea/genética , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Regiones Promotoras Genéticas , Radiografía , Transfección , Proteína Gli2 con Dedos de ZincRESUMEN
Transforming growth factor-beta (TGF-beta) signaling has been shown to promote invasion and metastasis in various models of human cancers. In this study, we investigated the efficacy of a TGF-beta type I receptor kinase inhibitor (TbetaRI-I) to limit early systemic metastases in an orthotopic xenograft model of lung metastasis and in an intracardiac injection model of experimental bone and lung metastasis using human breast carcinoma MDA-MB-435-F-L cells, a highly metastatic variant of human breast cancer MDA-MB-435 cells, expressing the enhanced green fluorescent protein (EGFP). Treatment of the cells with the TbetaRI-I had no effect on their growth but blocked TGF-beta-stimulated expression of integrin alpha(v)beta(3) and cell migration in vitro. Systemic administration of the TbetaRI-I via i.p. injection effectively reduced the number and size of the lung metastasis in both orthotopic xenograft and experimental metastasis models with no effects on primary tumor growth rate compared with controls. TbetaRI-I treatment also reduced the incidence of widespread early skeletal metastases in the femur, tibia, mandible, and spine detected by whole-body EGFP fluorescence imaging. Tumor burden in femora and tibiae was also reduced after TbetaRI-I treatment as detected by histomorphometry analysis compared with the placebo controls. Our results indicate for the first time that abrogation of TGF-beta signaling by systemic administration of the TbetaRI-I can inhibit both early lung and bone metastasis in animal model systems and suggest antimetastatic therapeutic potential of the TbetaRI-I.
Asunto(s)
Receptores de Activinas Tipo I/antagonistas & inhibidores , Neoplasias Óseas/prevención & control , Neoplasias de la Mama/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Neoplasias Pulmonares/prevención & control , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Animales , Neoplasias Óseas/enzimología , Neoplasias Óseas/secundario , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/secundario , Ratones , Proteínas Serina-Treonina Quinasas , Receptor Tipo I de Factor de Crecimiento Transformador beta , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Supporting roles of stromal cells in preferential colonization of myeloma cells in bone marrow and development of associated osteoclastic osteolysis through cell-cell interactions have been indicated. Here we examined the effects of a monoclonal antibody to alpha4 integrin (anti-alpha4 Ab) that disrupts myeloma cell-stromal cell interactions mediated via alpha4beta1 integrin and vascular cell adhesion molecule-1 (VCAM-1) on myeloma cell growth in bone marrow and accompanying osteolysis. The anti-alpha4 Ab decreased VCAM-1-stimulated 5TGM1/luc cell growth in culture. The 5TGM1 murine myeloma cells stably transfected with the firefly luciferase (5TGM1/luc) were inoculated from tail vein in bg/xid/nd mice. Preventative administration of the anti-alpha4 Ab suppressed the elevation of serum IgG2b levels, decreased 5TGM1/luc tumor burden with increased apoptosis in bone and spleen, reduced bone destruction with diminished number of osteoclasts, and prolonged survival of 5TGM1/luc-bearing mice. In contrast, therapeutic administration of the antibody failed to show these effects. However, therapeutic administration of the antibody combined with melphalan significantly suppressed serum IgG2b levels and tumor burden in bone. Our results suggest that the interactions with stromal cells via alpha4beta1/VCAM-1 are critical to the development of myeloma and associated osteolysis and that disruption of these interactions using anti-alpha4 Ab is a potential therapeutic approach for myeloma.
Asunto(s)
Anticuerpos/metabolismo , Integrina alfa4/inmunología , Mieloma Múltiple/inmunología , Osteoclastos/inmunología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Animales , Anticuerpos/química , Anticuerpos Monoclonales/química , Apoptosis , Células de la Médula Ósea/metabolismo , Huesos/patología , Comunicación Celular , División Celular , Línea Celular Tumoral , Supervivencia Celular , Inmunoglobulina G/sangre , Inmunoglobulina G/química , Integrina alfa4/química , Integrina alfa4beta1/metabolismo , Luciferasas/metabolismo , Melfalán/farmacología , Ratones , Osteoclastos/metabolismo , Osteólisis , Proteínas Recombinantes/química , Bazo/metabolismo , Bazo/patología , Factores de TiempoRESUMEN
Interleukin (IL)-1 is implicated in postmenopausal- and inflammation-mediated bone loss. Its expression is regulated by NF-kappaB and vice versa. To examine the role of NF-kappaB p50 and p52 (they are required for osteoclast formation during embryonic development) in IL-1-induced resorption, we used various NF-kappaB knockout (KO) mice, including p50-/- and p52-/- single KO, p50-/- and p52+/- (3/4KO), and p50-/- and p52-/- double KO (dKO) mice. IL-1 increased blood calcium and bone resorption in wild-type (wt), p50, and p52 single KO mice, but not in 3/4KO or dKO mice. Osteoclast formation was impaired in bone marrow cultures from 3/4KO compared with single KO and wt mice treated with IL-1. IL-1 receptor expression was similar in colony forming unit-granulocyte macrophage (CFU-GM) colony cells from wt and dKO mice. However, IL-1 promoted CFU-GM colony formation and survival as well as the formation, activity, and survival of osteoclasts generated from these colonies from wt mouse splenocytes, but not from dKO splenocytes. No difference in expression of the osteoclast regulatory cytokines, RANKL, and OPG, was observed in osteoblasts from wt and dKO mice. Thus, expression of either NF-kappaB p50 or p52 is required in osteoclasts and their precursors, rather than osteoblasts, for IL-1-mediated bone resorption.