RESUMEN
Current osteosarcoma therapies cause severe treatment-related side effects and chemoresistance, and have low success rates. Consequently, alternative treatment options are urgently needed. Photodynamic therapy (PDT) is a minimally invasive, local therapy with proven clinical efficacy for a variety of tumor types. PDT is cytotoxic, provokes anti-vascular effects and stimulates tumor cell targeting mechanisms of the immune system and, consequently, has potential as a novel therapy for osteosarcoma patients. This study investigated the uptake and the dark- and phototoxicity and cytotoxic mechanisms of the photosensitizer (PS) 5,10,15,20-tetrakis(meta-hydroxyphenyl) chlorine (mTHPC, Foscan) and a liposomal mTHPC formulation (Foslip) in the human 143B and a mouse K7M2-derived osteosaroma cell line (K7M2L2) in vitro. Second, the tumor- and metastasis-suppressive efficacies of mTHPC formulations based PDT and associated mechanisms in intratibial, metastasizing osteosarcoma mouse models (143B/SCID and syngeneic K7M2L2/BALB/c) were studied. The uptake of Foscan and Foslip in vitro was time- and dose-dependent and resulted in mTHPC and light dose-dependent phototoxicity associated with apoptosis. In vivo, the uptake of both i.v. administered mTHPC formulations was higher in tumor than in healthy control tissue. PDT caused significant (Foscan p < 0.05, Foslip p < 0.001) tumor growth inhibition in both models. A significant (Foscan p < 0.001, Foslip p < 0.001) immune system-dependent suppression of lung metastasis was only observed in the K7M2L2/BALB/c model and was associated with a marked infiltration of T-lymphocytes at the primary tumor site. In conclusion, mTHPC-based PDT is effective in clinically relevant experimental osteosarcoma and suppresses lung metastasis in immunocompetent mice with beneficial effects of the liposomal mTHPC formulation Foslip.
Asunto(s)
Neoplasias Óseas/tratamiento farmacológico , Mesoporfirinas/uso terapéutico , Osteosarcoma/tratamiento farmacológico , Fotoquimioterapia , Animales , Apoptosis , Neoplasias Óseas/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Sistema Inmunológico , Liposomas/química , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Microscopía Confocal , Metástasis de la Neoplasia , Trasplante de Neoplasias , Osteosarcoma/metabolismo , Fármacos Fotosensibilizantes/uso terapéutico , Tibia/patologíaRESUMEN
p63 is a crucial regulator of epidermal development, but its transcriptional control has remained elusive. Here, we report the identification of a long-range enhancer (p63LRE) that is composed of two evolutionary conserved modules (C38 and C40), acting in concert to control tissue- and layer-specific expression of the p63 gene. Both modules are in an open and active chromatin state in human and mouse keratinocytes and in embryonic epidermis, and are strongly bound by p63. p63LRE activity is dependent on p63 expression in embryonic skin, and also in the commitment of human induced pluripotent stem cells toward an epithelial cell fate. A search for other transcription factors involved in p63LRE regulation revealed that the CAAT enhancer binding proteins Cebpa and Cebpb and the POU domain-containing protein Pou3f1 repress p63 expression during keratinocyte differentiation by binding the p63LRE enhancer. Collectively, our data indicate that p63LRE is composed of additive and partly redundant enhancer modules that act to direct robust p63 expression selectively in the basal layer of the epidermis.
Asunto(s)
Elementos de Facilitación Genéticos , Epidermis/embriología , Epidermis/metabolismo , Regulación del Desarrollo de la Expresión Génica , Queratinocitos/metabolismo , Fosfoproteínas/genética , Transactivadores/genética , Animales , Diferenciación Celular/genética , Células Cultivadas , Humanos , Queratinocitos/citología , Ratones Endogámicos C57BL , Morfogénesis/genética , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment. Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response(1). Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)(2). The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis. In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.
Asunto(s)
Neoplasias Óseas/tratamiento farmacológico , Osteosarcoma/tratamiento farmacológico , Fotoquimioterapia/métodos , Animales , Línea Celular Tumoral , Humanos , Mesoporfirinas/farmacocinética , Mesoporfirinas/farmacología , Ratones , Ratones SCID , Fármacos Fotosensibilizantes/farmacocinética , Fármacos Fotosensibilizantes/farmacologíaRESUMEN
Current combined surgical and neo-adjuvant chemotherapy of primary metastatic osteosarcoma (OS) is ineffective, reflected by a 5-year survival rate of affected patients of less than 20 %. Studies in experimental OS metastasis models pointed to the CXCR4/CXCL12 homing axis as a novel target for OS metastasis-suppressive treatment. The present study investigated for the first time the CXCR4-blocking principle in a spontaneously metastasizing human 143B OS cell line-derived orthotopic xenograft mouse model. The highly metastatic 143B cells, unlike the parental non-metastatic HOS cells, express functional CXCR4 receptors at the cell surface, as revealed in this study by RT/PCR of gene transcripts, by FACS analysis with the monoclonal anti CXCR4 antibody 12G5 (mAb 12G5) and by CXCL12 time- and dose-dependent stimulation of AKT and ERK phosphorylation. A significantly (p < 0.05) higher CXCL12 dose-dependent chemotactic response of 143B compared to HOS cells in a Boyden chamber trans-well migration assay suggested a crucial role of the CXCL12/CXCR4 homing axis in 143B cell lung metastasis. Repetitive treatment of mice with 143B cell-derived intratibial tumors given intravenous bolus injections of mAb12G5 indeed inhibited significantly (p < 0.01) the number of X-gal-stainable lung micrometastases of lacZ-transduced 143B cells. Antibody treatment had also a mild inhibitory effect on primary tumor growth associated with remarkably less osteolysis, but it did not affect the number of developing lung macrometastases. In conclusion, these results demonstrate considerable potential of high-affinity CXCR4-blocking agents for OS tumor cell homing suppressive treatment in metastasizing OS complementary to current (neo)-adjuvant chemotherapy.
Asunto(s)
Anticuerpos/administración & dosificación , Neoplasias Pulmonares/secundario , Metástasis de la Neoplasia/tratamiento farmacológico , Receptores CXCR4/administración & dosificación , Animales , Anticuerpos/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Inmunoterapia , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Ratones , Metástasis de la Neoplasia/inmunología , Metástasis de la Neoplasia/patología , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/inmunología , Osteosarcoma/patología , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/inmunología , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
More effective treatment of metastasizing osteosarcoma with a current mean 5-year survival rate of less than 20% requires more detailed knowledge on mechanisms and key regulatory molecules of the complex metastatic process. CXCR4, the receptor of the chemokine CXCL12, has been reported to promote tumor progression and metastasis in osteosarcoma. CXCR7 is a recently deorphanized CXCL12-scavenging receptor with so far not well-defined functions in tumor biology. The present study focused on a potential malignancy enhancing function of CXCR7 in interaction with CXCR4 in osteosarcoma, which was investigated in an intratibial osteosarcoma model in SCID mice, making use of the human 143B osteosarcoma cell line that spontaneously metastasizes to the lung and expresses endogenous CXCR4. 143B osteosarcoma cells stably expressing LacZ (143B-LacZ cells) were retrovirally transduced with a gene encoding HA-tagged CXCR7 (143B-LacZ-X7-HA cells). 143B-LacZ-X7-HA cells co-expressing CXCR7 and CXCR4 exhibited CXCL12 scavenging and enhanced adhesion to IL-1ß-activated HUVEC cells compared to 143B-LacZ cells expressing CXCR4 alone. SCID mice intratibially injected with 143B-LacZ-X7-HA cells had significantly (p<0.05) smaller primary tumors, but significantly (p<0.05) higher numbers of lung metastases than mice injected with 143B-LacZ cells. Unexpectedly, 143B-LacZ-X7-HA cells, unlike 143B-LacZ cells, also metastasized with high incidence to the auriculum cordis. In conclusion, expression of the CXCL12 scavenging receptor CXCR7 in the CXCR4-expressing human 143B osteosarcoma cell line enhances its metastatic activity in intratibial primary tumors in SCID mice that predominantly metastasize to the lung and thereby closely mimic the human disease. These findings point to CXCR7 as a target, complementary to previously proposed CXCR4, for more effective metastasis-suppressive treatment in osteosarcoma.
Asunto(s)
Neoplasias Óseas/genética , Neoplasias Óseas/patología , Neoplasias Pulmonares/secundario , Osteosarcoma/genética , Osteosarcoma/patología , Receptores CXCR4/genética , Receptores CXCR/genética , Animales , Neoplasias Óseas/metabolismo , Adhesión Celular/genética , Línea Celular Tumoral , Quimiocina CXCL12/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones SCID , Osteosarcoma/metabolismo , Receptores CXCR/metabolismo , Receptores CXCR4/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
UNLABELLED: The aim of this study was to characterize the different phenotypes of osteosarcoma by PET, comparing the uptake of 3 tracers ((18)F-FDG, (18)F-fluoromisonidazole [(18)F-FMISO], and (18)F-fluoride) in preclinical mouse models that reflect the heterogeneity of the human disease. METHODS: Mouse LM8 osteosarcoma, human 143B, and Caprin-1 stably overexpressing SaOS-2 cells were injected intratibially in C3H and severe-combined immunodeficient mice. PET imaging with (18)F-FDG, (18)F-FMISO, and (18)F-fluoride was performed in these mouse models, and a ratio between the standardized uptake value of the primary tumor and a control area of bone was calculated and compared among the models. Histology and immunohistochemistry were performed to confirm the PET findings. RESULTS: The pattern of tracer uptake differed among the primary tumors of the 3 models in accordance with the histology and immunohistochemistry on primary tumor sections. The osteolytic tumors in the 143B model showed the highest uptake of (18)F-FDG, an indicator of glucose metabolism, which was significantly higher (P < 0.05) than in the SaOS-2/Caprin-1 model and correlated with the percentage of Ki67-positive cells in the primary tumors. Hypoxia, indicated by (18)F-FMISO accumulation, was higher in the SaOS-2/Caprin-1 and 143B cell line-derived tumors (P < 0.01). Finally (18)F-fluoride, a marker of bone remodeling, correlated with the osteoblastic phenotype. The SaOS-2/Caprin-1 cell-derived tumors showed a significantly higher uptake than the moderately osteoblastic LM8 (P < 0.05) and the osteolytic 143B (P < 0.01) cell line-derived tumors. CONCLUSION: Differential PET imaging with tracers indicating metabolic activity, hypoxia, or bone remodeling will be helpful for the characterization of different osteosarcoma phenotypes and subsequent evaluation of more specific treatment modalities targeting the processes that are predominant in each specific tumor type or subtype.
Asunto(s)
Osteosarcoma/diagnóstico por imagen , Fenotipo , Tomografía de Emisión de Positrones , Animales , Neoplasias Óseas/diagnóstico por imagen , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Femenino , Fluoruros , Fluorodesoxiglucosa F18 , Humanos , Ratones , Misonidazol/análogos & derivados , Osteoblastos/patología , Osteoclastos/patología , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/metabolismo , Osteosarcoma/patología , Tibia/patologíaRESUMEN
Osteosarcoma (OS) is the most frequent primary malignant bone cancer in children and adolescents with a high propensity for lung metastasis. Therefore, it is of great importance to identify molecular markers leading to increased metastatic potential in order to devise more effective therapeutic strategies that suppress metastasis, the major cause of death in OS. CD44, the principal receptor for the extracellular matrix component hyaluronan (HA), is frequently found overexpressed in tumor cells and has been implicated in metastatic spread in various cancer types. Here, we investigated the effects of stable shRNA-mediated silencing of CD44 gene products on in vitro and in vivo metastatic properties of the highly metastatic human 143-B OS cell line. In vitro, CD44 knockdown resulted in a 73% decrease in the adhesion to HA, a 57% decrease in the migration rate in a trans-filter migration assay, and a 28% decrease in the cells' capacity for anchorage-independent growth in soft agar compared to the control cells, implicating that CD44 expression contributes to the metastatic activity of 143-B cells. However, making use of an orthotopic xenograft OS mouse model, we demonstrated that reduced CD44 expression facilitated primary tumor growth and formation of pulmonary metastases. The enhanced malignant phenotype was associated with decreased adhesion to HA and reduced expression of the tumor suppressor merlin in vivo. In conclusion, our study identified CD44 as a metastasis suppressor in this particular experimental OS model.
Asunto(s)
Neoplasias Óseas/genética , Neoplasias Óseas/patología , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Receptores de Hialuranos/genética , Osteosarcoma/genética , Osteosarcoma/patología , Animales , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Humanos , Receptores de Hialuranos/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Ratones , Ratones SCID , Metástasis de la Neoplasia , Interferencia de ARN , Trasplante Heterólogo , Carga Tumoral/genéticaRESUMEN
BACKGROUND: Osteosarcoma is the most common malignant bone tumor in children and young adults. Since the introduction of chemotherapy, the 5-year survival rate of patients with non-metastatic osteosarcoma is ~70%. The main problems in osteosarcoma therapy are the occurrence of metastases, severe side-effects and chemoresistance. Antiproliferative and apoptotic effects of quercetin were shown in several types of cancers, including breast cancer and lung carcinoma. MATERIALS AND METHODS: The present study investigates the cytotoxic potential of quercetin, a dietary flavonoid, in a highly metastasizing human osteosarcoma cell line, 143B. RESULTS: We found that quercetin induces growth inhibition, G2/M phase arrest, and apoptosis in the 143B osteosarcoma cell line. We also observed impaired adhesion and migratory potential after the addition of quercetin. CONCLUSION: Since quercetin has already been shown to have low side effects in a clinical phase I trial in advanced cancer patients, this compound may have considerable potential for osteosarcoma treatment.
Asunto(s)
Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Neoplasias Óseas/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Osteosarcoma/tratamiento farmacológico , Quercetina/farmacología , Western Blotting , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Adhesión Celular/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Citometría de Flujo , Humanos , Osteosarcoma/metabolismo , Osteosarcoma/patología , Células Tumorales Cultivadas , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Formation of metastases in the lungs is the major cause of death in patients suffering from osteosarcoma (OS). Metastases at presentation and poor response to preoperative chemotherapy are strong predictors for poor patient outcome. The elucidation of molecular markers that promote metastasis formation and/or chemoresistance is therefore of importance. CD44 is a plasma membrane glycoprotein that binds to the extracellular matrix component hyaluronan (HA) and has been shown to be involved in metastasis formation in a variety of other tumors. Here we investigated the role of CD44 expression on OS tumor formation and metastasis. High CD44 expression, evaluated with a tissue microarray including samples from 53 OS patients and stained with a pan-CD44 antibody (Hermes3), showed a tendency (p < 0.08) to shortened overall survival. However, nonresponders and patients with lung metastases and high CD44 expression had significantly poorer prognosis than patients with low CD44 expression. Overexpression of the standard CD44 isoform (CD44s) and its HA-binding defective mutant R41A in osteoblastic SaOS-2 cells resulted in HA-independent higher migration rates and increased chemoresistance, partially dependent on HA. In an orthotopic mouse model of OS, overexpression of CD44s in SaOS-2 cells resulted in an HA-dependent increased primary tumor formation and increased numbers of micrometastases and macrometastases in the lungs. In conclusion, although CD44 failed to be an independent predictor for patient outcome in this limited cohort of OS patients, increased CD44 expression was associated with even worse survival in patients with chemoresistance and with lung metastases. CD44-associated chemoresistance was also observed in vitro, and increased formation of lung metastases was found in vivo in SCID mice.
Asunto(s)
Carcinogénesis/metabolismo , Receptores de Hialuranos/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Osteosarcoma/metabolismo , Osteosarcoma/patología , Adolescente , Adulto , Animales , Carcinogénesis/patología , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Niño , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Ácido Hialurónico/farmacología , Estimación de Kaplan-Meier , Masculino , Ratones SCID , Persona de Mediana Edad , Pronóstico , Tibia/efectos de los fármacos , Tibia/patología , Resultado del Tratamiento , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto JovenRESUMEN
Photodynamic therapy (PDT) is a minimally invasive therapeutic modality approved for palliative and curative treatment of some forms of local cancers, precancerous lesions and nononcological disorders. As a prerequisite for future studies in animal models aiming at an intraoperative application of PDT in osteosarcoma (OS), in the present study, we investigated the uptake and the dark- and photo-toxicity of the photosensitizer mTHPC in the metastatic human OS cell line 143B, which, intratibially injected into SCID mice, reproduces spontaneous, aggressive lung metastasis, the main cause of death in OS patients. The uptake of mTHPC by 143B cells was time- and dose-dependent. mTHPC accumulated to higher levels in the 143B than in the parental low-metastatic HOS cell line. A significant decrease in viability of 143B cells, reflecting mTHPC dark-toxicity, occurred upon incubation in the dark at mTHPC concentrations ≥2.5 µg mL(-1). In phototoxicity experiments with illumination by 652 nm laser light (2.5-10 J cm(-2)), the half-maximal lethal doses of mTHPC ranged from 0.012 to 0.047 µg mL(-1). This treatment activated caspase-3, -7 and -9 and Z-VAD-FMK-inhibitable PARP cleavage, indicating caspase-dependent apoptosis. In conclusion, PDT with mTHPC is effective in the metastatic 143B human osteosarcoma cell line in vitro.