RESUMEN
Neuroblastoma is a pediatric cancer with significant clinical heterogeneity. Despite extensive efforts, it is still difficult to cure children with high-risk neuroblastoma. Immunotherapy is a promising approach to treat children with this devastating disease. We have previously reported that macrophages are important effector cells in high-risk neuroblastoma. In this perspective article, we discuss the potential function of the macrophage inhibitory receptor SIRPA in the homeostasis of tumor-associated macrophages in high-risk neuroblastoma. The ligand of SIRPA is CD47, known as a "don't eat me" signal, which is highly expressed on cancer cells compared to normal cells. CD47 is expressed on both tumor and stroma cells, whereas SIRPA expression is restricted to macrophages in high-risk neuroblastoma tissues. Notably, high SIRPA expression is associated with better disease outcome. According to the current paradigm, the interaction between CD47 on tumor cells and SIRPA on macrophages leads to the inhibition of tumor phagocytosis. However, data from recent clinical trials have called into question the use of anti-CD47 antibodies for the treatment of adult and pediatric cancers. The restricted expression of SIRPA on macrophages in many tissues argues for targeting SIRPA on macrophages rather than CD47 in CD47/SIRPA blockade therapy. Based on the data available to date, we propose that disruption of the CD47-SIRPA interaction by anti-CD47 antibody would shift the macrophage polarization status from M1 to M2, which is inferred from the 1998 study by Timms et al. In contrast, the anti-SIRPA F(ab')2 lacking Fc binds to SIRPA on the macrophage, mimics the CD47-SIRPA interaction, and thus maintains M1 polarization. Anti-SIRPA F(ab')2 also prevents the binding of CD47 to SIRPA, thereby blocking the "don't eat me" signal. The addition of tumor-opsonizing and macrophage-activating antibodies is expected to enhance active tumor phagocytosis.
Asunto(s)
Antígenos de Diferenciación , Antígeno CD47 , Neuroblastoma , Receptores Inmunológicos , Antígeno CD47/metabolismo , Humanos , Receptores Inmunológicos/metabolismo , Macrófagos/metabolismoRESUMEN
Neuroblastoma is the most common extracranial childhood solid tumor. The majority of high-risk neuroblastoma is resistant/refractory to the current high intensity therapy. Neuroblastoma lacks classical HLA Class I expression and exhibits low mutation burden, allowing neuroblastoma cells to evade CD8+ T cell-mediated immunity. Neuroblastoma cells do not express PD-L1, and tumor-associated macrophages are the predominant PD-L1+ cells in the tumor. In this study, we performed gene expression profiling and survival analyses on large neuroblastoma datasets to address the prognostic effect of PD-L1 gene expression and the possible involvement of the SLAMF7 pathway in the anti-neuroblastoma immunity. High-level expression of PD-L1 was found significantly associated with better outcome of high-risk neuroblastoma patients; two populations of PD-1+ PD-L1+ macrophages could be present in high-risk tumors with PD-1/PD-L1 ratios, ≈1 and >1. Patients with the PD-1/PD-L1 ratio >1 tumor showed inferior survival. High-level co-expression of SLAMF7 and SH2D1B was significantly associated with better survival of the high-risk neuroblastoma patients. Together, this study supports the hypothesis that macrophages are important effector cells in the anti-high-risk neuroblastoma immunity, that PD-1 blockade therapy can be beneficial to the high-risk neuroblastoma subset with the PD-1/PD-L1 expression ratio >1, and that SLAMF7 is a new therapeutic target of high-risk neuroblastoma.
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Antígeno B7-H1 , Macrófagos , Neuroblastoma , Antígeno B7-H1/genética , Linfocitos T CD8-positivos , Humanos , Macrófagos/inmunología , Neuroblastoma/genética , Neuroblastoma/inmunología , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/metabolismo , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/genética , Escape del TumorRESUMEN
Peripheral neuroblastic tumors (neuroblastoma, ganglioneuroblastoma and ganglioneuroma) are heterogeneous and their diverse and wide range of clinical behaviors (spontaneous regression, tumor maturation and aggressive progression) are closely associated with genetic/molecular properties of the individual tumors. The International Neuroblastoma Pathology Classification, a biologically relevant and prognostically significant morphology classification distinguishing the favorable histology (FH) and unfavorable histology (UH) groups in this disease, predicts survival probabilities of the patients with the highest hazard ratio. The recent advance of neuroblastoma research with precision medicine approaches demonstrates that tumors in the UH group are also heterogeneous and four distinct subgroups-MYC, TERT, ALT and null-are identified. Among them, the first three subgroups are collectively named extremely unfavorable histology (EUH) tumors because of their highly aggressive clinical behavior. As indicated by their names, these EUH tumors are individually defined by their potential targets detected molecularly and immunohistochemically, such as MYC-family protein overexpression, TERT overexpression and ATRX (or DAXX) loss. In the latter half on this paper, the current status of therapeutic targeting of these EUH tumors is discussed for the future development of effective treatments of the patients.
Asunto(s)
Biomarcadores de Tumor , Ganglioneuroblastoma , Ganglioneuroma , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias , Medicina de Precisión , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Ganglioneuroblastoma/genética , Ganglioneuroblastoma/metabolismo , Ganglioneuroblastoma/patología , Ganglioneuroblastoma/terapia , Ganglioneuroma/genética , Ganglioneuroma/metabolismo , Ganglioneuroma/patología , Ganglioneuroma/terapia , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estadificación de NeoplasiasRESUMEN
Neuroblastoma is the most common extracranial childhood solid tumor. The majority of high-risk neuroblastoma is resistant/refractory to the current high intensity therapy, and the survival of these patients remains poor for the last three decades. To effectively treat these extremely unfavorable neuroblastomas, innovative immunotherapy approaches would be the most promising. In this article, we discuss the identity of tumor-infiltrating effector cells and immunosuppressive cells in high-risk neuroblastoma. Neuroblastoma is unique in that it expresses little or no classical HLA Class I and II. In contrast, high-risk neuroblastomas express the stress-responsive non-classical Class I, HLA-E molecule. HLA-E is the ligand of activating receptors NKG2C/E that are expressed on memory NK cells, CD8+T cells and CD4 CTLs. By examining a comprehensive RNA-seq gene expression dataset, we detected relatively high levels of CD4 expression in high-risk neuroblastoma tissues. The majority of CD4+ cells were CD3+, and thus they were likely tumor-associated CD4+T cells. In addition, high-level of both CD4 and NKG2C/E expression was associated with prolonged survival of the high-risk neuroblastoma patients, but CD8 levels were not, further suggesting that the CD4+ NKG2C/E+ T cells or CD4 CTL conferred cytotoxicity against the neuroblastoma cells. However, this T cell mediated- "protective effect" declined over time, in part due to the progressive formation of immunosuppressive tumor microenvironment. These observations suggest that to improve survival of high-risk neuroblastoma patients, it is essential to gain insights into how to enhance CD4 CTL cytotoxicity and control the immunosuppressive tumor microenvironment during the course of the disease.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Citotoxicidad Inmunológica/inmunología , Neuroblastoma/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Niño , Humanos , Inmunoterapia , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Subfamília C de Receptores Similares a Lectina de Células NK/inmunología , Subfamília C de Receptores Similares a Lectina de Células NK/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/terapia , Factores de Riesgo , Linfocitos T Citotóxicos/metabolismo , Microambiente Tumoral/inmunologíaRESUMEN
Stage 4S neuroblastoma (4SNB) is associated with spontaneous tumor regression and an excellent prognosis. However, a small group of the patients have a poor prognosis. One hundred eighty-five stage 4SNB cases filed at the Children's Oncology Group Neuroblastoma Pathology Reference Laboratory were studied. MYCN oncogene status [non-amplified (NA) vs. Amplified (A)] determined by fluorescence in situ hybridization, MYC-family (MYCN/MYC) protein expression [no-overexpression(-)/(+/-) vs. overexpression(+)] by immunohistochemistry and histopathology by International Neuroblastoma Pathology Classification [Favorable Histology (FH) vs. Unfavorable Histology (UH)] with particular attention to nucleolar hypertrophy [NH(-) vs. (+)] were assessed with patient survival. One hundred forty-seven (79.5%) tumors were MYCN-NA, FH, MYC-family protein(-)/(+/-), and NH(-) with a good prognosis [88.5±3.1% 3-y event-free survival (EFS); 94.1±2.3% 3-y overall survival (OS)]. Among MYCN-NA tumors, 11 demonstrated MYCN protein(+) with a moderate and uniform (M/U) staining pattern: they were FH(10/11), NH(-), 1 showed MYC protein(+) simultaneously, and all patients are alive. Also found were 5 MYC protein(+) and MYCN(-)/(+/-) tumors; they were FH without NH (4/5), and all patients are alive. Among MYCN-A tumors, 18 had MYCN protein(+) with a strong and heterogeneous (S/H) staining pattern, 9 had UH (44.4±23.4% EFS/OS) and 9 had FH (68.6±19.2% EFS/OS), and 15 showed NH(+). Two tumors had MYCN protein(-)/(+/-) despite MYCN-A; both were FH and NH(-), and 1 patient died. S/H staining pattern of MYCN protein overexpression by immunohistochemistry was associated with MYCN amplification, NH(+) and a poor prognosis. In contrast, the M/U staining pattern was associated with MYCN nonamplification and NH(-), and had no adverse prognostic effects for the stage 4SNB patients.
Asunto(s)
Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/patología , Biomarcadores de Tumor/genética , Femenino , Amplificación de Genes , Humanos , Inmunohistoquímica , Lactante , Recién Nacido , Masculino , Estadificación de Neoplasias , PronósticoRESUMEN
Neuroblastoma (NB) tumor substantially contributes to childhood cancer mortality. The design of novel drugs targeted to specific molecular alterations becomes mandatory, especially for high-risk patients burdened by chemoresistant relapse. The dysregulated expression of MYCN, ALK, and LIN28B and the diminished levels of miR-34a and let-7b are oncogenic in NB. Due to the ability of miRNA-mimics to recover the tumor suppression functions of miRNAs underexpressed into cancer cells, safe and efficient nanocarriers selectively targeted to NB cells and tested in clinically relevant mouse models are developed. The technology exploits the nucleic acids negative charges to build coated-cationic liposomes, then functionalized with antibodies against GD2 receptor. The replenishment of miR-34a and let-7b by NB-targeted nanoparticles, individually and more powerfully in combination, significantly reduces cell division, proliferation, neoangiogenesis, tumor growth and burden, and induces apoptosis in orthotopic xenografts and improves mice survival in pseudometastatic models. These functional effects highlight a cooperative down-modulation of MYCN and its down-stream targets, ALK and LIN28B, exerted by miR-34a and let-7b that reactivate regulatory networks leading to a favorable therapeutic response. These findings demonstrate a promising therapeutic efficacy of miR-34a and let-7b combined replacement and support its clinical application as adjuvant therapy for high-risk NB patients.
Asunto(s)
MicroARNs , Nanopartículas , Neuroblastoma , Animales , Línea Celular Tumoral , Proliferación Celular , Niño , Humanos , Ratones , MicroARNs/genética , Recurrencia Local de Neoplasia , Proteínas de Unión al ARNRESUMEN
BACKGROUND: Neuroblastoma is the most common pediatric extracranial solid malignancy with limited effective treatment. We have shown that sustained-release, single drugs delivered locally through a silk-based biomaterial are effective in decreasing orthotopic neuroblastoma xenograft growth. We further optimized this approach and hypothesized that increasing doses of local chemotherapy or delivering 2 chemotherapeutic agents simultaneously inhibit additional tumor growth. METHODS: MYCN-amplified and non-MYCN-amplified neuroblastoma cells were treated with combinations of cisplatin, vincristine, doxorubicin, and etoposide to determine cytotoxicity and synergy. Drug-loaded silk material was created, and the amounts of drug released from the material over time were recorded. Murine orthotopic neuroblastoma xenografts were generated; tumors were implanted with single- or dual-agent chemotherapy-loaded silk. Ultrasound was used to monitor tumor growth, and tumor histology was evaluated. RESULTS: In vitro, vincristine/cisplatin combination was synergistic and significantly decreased cell viability relative to other combinations. Both drugs loaded into silk could be released effectively for over 2 weeks. Locally implanted vincristine/cisplatin silk induced increased tumor growth suppression compared with either agent alone in MYCN-amplified tumors (P < .05). The dose-dependent effect seen in MYCN-amplified tumors treated with combination therapy diminished at higher doses in non-MYCN-amplified tumors, with little benefit with doses >50 µg to 500 µg for vincristine-cisplatin, respectively. Tumor histology demonstrated tumor cell necrosis adjacent to drug-loaded silk material and presence of large cell neuroblastoma. CONCLUSION: Local delivery of sustained release chemotherapy can suppress tumor growth especially at high doses or with 2 synergistic drugs. Locally delivered dual therapy is a promising approach for future clinical testing.
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Cisplatino/administración & dosificación , Doxorrubicina/administración & dosificación , Etopósido/administración & dosificación , Neuroblastoma/tratamiento farmacológico , Vincristina/administración & dosificación , Animales , Preparaciones de Acción Retardada , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Sistemas de Liberación de Medicamentos , Humanos , Ratones , Trasplante de Neoplasias , Neuroblastoma/patología , Seda , Células Tumorales CultivadasRESUMEN
Immunotherapy targeting GD2 is a primary treatment for patients with high-risk neuroblastoma. Dinutuximab is a monoclonal antibody with great clinical promise but is limited by side effects such as severe pain. Local delivery has emerged as a potential mechanism to deliver higher doses of therapeutics into the tumor bed, while limiting systemic toxicity. We aim to deliver dinutuximab locally in a lyophilized silk fibroin foam for the treatment of an orthotopic neuroblastoma mouse model. Dinutuximab-loaded silk fibroin foams were fabricated through lyophilization. In vitro release profile and bioactivity of the release through complement-dependent cytotoxicity were characterized. MYCN-amplified neuroblastoma cells (KELLY) were injected into the left gland of mice to generate an orthotopic neuroblastoma model. Once the tumor volume reached 100 mm3 , dinutuximab-, human IgG-, or buffer-loaded foams were implanted into the tumor and growth was monitored using high-resolution ultrasound. Post-resection histology was performed on tumors. Dinutuximab-loaded silk fibroin foams exhibited a burst release, with slow release thereafter in vitro with maintenance of bioactivity. The dinutuximab-loaded foam significantly inhibited xenograft tumor growth compared to IgG- and buffer-loaded foams. Histological analysis revealed the presence of dinutuximab within the tumor and neutrophils and macrophages infiltrating into dinutuximab-loaded silk foam. Tumors treated with local dinutuximab had decreased MYCN expression on histology compared to control or IgG-treated tumors. Silk fibroin foams offer a mechanism for local release of dinutuximab within the neuroblastoma tumor. This local delivery achieved a significant decrease in tumor growth rate in a mouse orthotopic tumor model.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Fibroínas/química , Neuroblastoma/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/química , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Apoptosis , Proliferación Celular , Femenino , Liofilización , Humanos , Ratones , Ratones Desnudos , Neuroblastoma/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Large cell neuroblastomas (LCN) are frequently seen in recurrent, high-risk neuroblastoma but are rare in primary tumors. LCN, characterized by large nuclei with prominent nucleoli, predict a poor prognosis. We hypothesize that LCN can be created with high-dose intra-tumoral chemotherapy and identified by a digital analysis system. METHODS: Orthotopic mouse xenografts were created using human neuroblastoma and treated with high-dose chemotherapy delivered locally via sustained-release silk platforms, inducing tumor remission. After recurrence, LCN populations were identified on H&E sections manually. Clusters of typical LCN and non-LCN cells were divided equally into training and test sets for digital analysis. Marker-controlled watershed segmentation was used to identify nuclei and characterize their features. Logistic regression was developed to distinguish LCN from non-LCN. RESULTS: Image analysis identified 15,000 nuclei and characterized 70 nuclear features. A 19-feature model provided AUC >0.90 and 100% accuracy when >30% nuclei/cluster were predicted as LCN. Overall accuracy was 87%. CONCLUSIONS: We recreated LCN using high-dose chemotherapy and developed an automated method for defining LCN histologically. Features in the model provide insight into LCN nuclear phenotypic changes that may be related to increased activity. This model could be adapted to identify LCN in human tumors and correlated with clinical outcomes.
Asunto(s)
Antineoplásicos , Interpretación de Imagen Asistida por Computador/métodos , Neuroblastoma , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Núcleo Celular/patología , Humanos , Inyecciones Intralesiones , Ratones , Neoplasias Experimentales/diagnóstico por imagen , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Neuroblastoma/clasificación , Neuroblastoma/diagnóstico por imagen , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
[This corrects the article DOI: 10.1021/acsmedchemlett.7b00126.].
RESUMEN
PURPOSE: MYCN oncogene amplification is an independent predictor of poor prognosis in neuroblastoma. CX-5461 is a small molecular inhibitor that prevents initiation of ribosomal RNA (rRNA) synthesis by RNA Pol I, down-regulating MYCN/MYC proteins. We hypothesize that neuroblastoma tumor growth can be suppressed by CX-5461. METHODS: MYCN-amplified (KELLY, IMR5) and nonamplified (SY5Y, SKNAS) neuroblastoma cells were treated with CX-5461. MYCN/MYC expression after 24-48 h was determined by Western blot. Orthotopic neuroblastoma tumors created in mice using KELLY cells were treated with CX-5461-loaded silk films implanted locally. Tumor growth was monitored using ultrasound. Histologic evaluation of tumors was performed. RESULTS: IC50 for KELLY, IMR5, SY5Y, and SKNAS cells to CX-5461 was 0.75 µM, 0.02 µM, 0.8 µM, and 1.7 µM, respectively. CX-5461 down-regulated MYCN and MYC proteins at 0.25-1.0 µM on Western blot analysis. CX-5461-loaded silk film released 23.7±3 µg of the drug in 24 h and 48.2±3.9 µg at 120 h. KELLY tumors treated with CX-5461-loaded film reached 800 mm3 after 7.8±1.4 days, while those treated with control film reached the same size on 5.1±0.6 days (p=0.03). CX-5461-treated tumors showed collapse of nucleolar hypertrophy and MYCN protein downregulation. CONCLUSION: We demonstrated that local delivery of CX-5461 via sustained release platform can suppress orthotopic neuroblastoma tumor growth, especially those with MYCN/MYC overexpression.
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Benzotiazoles/farmacología , Regulación hacia Abajo/efectos de los fármacos , Proteína Proto-Oncogénica N-Myc/metabolismo , Naftiridinas/farmacología , Neuroblastoma , Animales , Línea Celular Tumoral , Humanos , Ratones , Neuroblastoma/metabolismo , Neuroblastoma/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Neuroblastoma is the most common extracranial childhood tumor, and current treatment requires surgical resection and multidrug chemotherapy. Local, perioperative delivery of chemotherapeutics is a promising treatment method for solid tumors that require surgical removal. In this study, we have aimed to develop a controlled-release implant system to deliver cisplatin in tumor or tumor resection area. Silk fibroin, a biodegradable, nonimmunogenic biopolymer was used to encapsulate different doses of cisplatin in a reservoir system. The physical integrity of the reservoirs was characterized by evaluating the crystalline structure of silk secondary structure using FTIR spectroscopy. The in vitro release of cisplatin was evaluated in phosphate-buffered saline at 37°C, and the reservoirs were able to release the drug up to 30 days. The cytotoxicity of cisplatin and cisplatin reservoirs were tested on KELLY cells. Cytotoxicity data showed 3.2 µg/mL cisplatin was required to kill 50% of the cell population, and the released cisplatin from the silk reservoirs showed significant cytotoxicity up to 21 days. Intratumoral implantation of silk reservoirs into an orthotopic neuroblastoma mouse model decreased tumor growth significantly when compared with control subjects. These results suggest that silk reservoirs are promising carriers for cisplatin delivery to the tumor site.
Asunto(s)
Antineoplásicos/administración & dosificación , Cisplatino/administración & dosificación , Preparaciones de Acción Retardada/química , Fibroínas/química , Neuroblastoma/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Bombyx/química , Línea Celular Tumoral , Cisplatino/uso terapéutico , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Ratones , Ratones DesnudosRESUMEN
PURPOSE: Precise excision of neuroblastoma is challenging, especially when tumors adhere to vital structures. Indocyanine green (ICG), an FDA-approved dye with absorption peaking at 800â¯nm, can absorb the near IR laser energy and release heat in the dyed tissue. We hypothesize that by injecting ICG at tumor sites followed by precise laser application, tumor cell death can be selectively targeted. METHODS: Orthotopic neuroblastoma tumors were created in the adrenal gland of immunocompromised mice. Tumor, liver, kidney, and muscle tissues were chosen for ICG injection. Intervention variables included presence of tumor capsule, continuous vs. pulsed laser treatment and total energy delivered. Control groups included laser or ICG only. Tissues were stained with hematoxylin/eosin. RESULTS: Continuous wave laser generated excessive heat, causing damage in all tissues. When using pulsed laser treatment, liver, kidney, muscle, and intact tumor tissues showed no cell death when treated with laser alone or laser plus ICG. Tumor tissue with the capsule removed, however, showed cell death on histology. CONCLUSIONS: Pulsed laser treatment combined with ICG causes targeted tumor cell death in neuroblastoma tumor without capsule. No cell death was observed when tumor capsule was present, when only laser was used, or when applied over non-tumor tissues.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/terapia , Colorantes/farmacología , Verde de Indocianina/farmacología , Terapia por Láser/métodos , Neuroblastoma/terapia , Neoplasias de las Glándulas Suprarrenales/patología , Animales , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Terapia Combinada/métodos , Modelos Animales de Enfermedad , Femenino , Riñón/patología , Hígado/patología , Ratones , Músculos/patología , Neuroblastoma/patologíaRESUMEN
BACKGROUND: Advanced-stage neuroblastoma patients require multiagent chemotherapy. Intratumoral implantation of vincristine-loaded silk gel uses local diffusion to decrease orthotopic neuroblastoma tumor growth in mice. We hypothesize that injecting vincristine-loaded silk gel into 8 locations within the tumor, instead of only centrally, decreases the diffusion distance and improves tumor growth suppression. METHODS: Human neuroblastoma cells, KELLY, were injected into mouse adrenal glands to create orthotopic tumors. After the tumors reached 100 mm3 by ultrasound, silk gels loaded with 50 µg vincristine were injected centrally or in 8 areas throughout the tumor. Drug-release profile was measured in vitro. Endpoints were tumor size >1,000 mm3 and histologic examination. RESULTS: Vincristine-loaded silk gels suppressed tumor growth up to an inflection point (458.7 ± 234.4 mm3 for central, 514.3 ± 165.8 mm3 for 8-point injection) before tumor growth accelerated >200 mm3 over 3 days. The time to inflection point was 6.6 days for central, 13.3 days for 8-point injection (P < .05). Using the sphere volume equation to approximate tumor volume, splitting the volume into 1/8 decreased the diffusion radius by 1/2. Histologic examination confirmed tumor necrosis adjacent to vincristine-loaded silk gel. CONCLUSION: Injecting vincristine-loaded sustained release silk gel at 8 separate locations halved the diffusion distance and doubled the time for the tumor to reach the growth inflexion point.
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Antineoplásicos/administración & dosificación , Neuroblastoma/tratamiento farmacológico , Vincristina/administración & dosificación , Animales , Materiales Biocompatibles , Línea Celular Tumoral , Difusión , Sistemas de Liberación de Medicamentos , Geles , Humanos , Inyecciones Intralesiones , Ratones , Neuroblastoma/patología , Seda , Extractos de Tejidos , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
High-risk neuroblastoma requires surgical resection and multi-drug chemotherapy. This study aimed to develop an extended release, implantable and degradable delivery system for etoposide, commonly used for neuroblastoma treatment. Different concentrations of silk, a biodegradable, non-toxic, non-immunogenic material were employed to prepare etoposide-loaded wafer formulations. Secondary structure of silk in the formulations was characterized using Fourier Transform Infrared (FTIR) spectroscopy and optimized based on the crystalline structure. Accelerated in vitro degradation studies under different conditions such as acidic, alkaline, oxidizing mediums and high temperature, were performed. The integrity of the silk wafer structure was maintained unless exposed to 0.1â¯N NaOH for 24â¯h. In vitro release of etoposide was performed in PBS (phosphate buffered saline) at 37⯰C. Silk coated 6% wafers released the drug up to 45â¯days, while uncoated wafers released the drug for 30â¯days. Cytotoxicity study was performed on KELLY cells to evaluate the etoposide cytotoxicity (LC50) and the long-term efficacy of the etoposide wafer formulations. The results showed that etoposide killed 50% of the cells at 1⯵g/mL concentration and the wafer formulations demonstrated significant cytotoxicity up to 22â¯days when compared to untreated cells. Using an orthotopic neuroblastoma mouse model, intra-tumoral implantation of the coated 6%, uncoated 6%, or uncoated 3% silk wafers were all effective at decreasing tumor growth. Histological examination revealed tumor cell necrosis adjacent to the drug-loaded silk wafer.
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Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/uso terapéutico , Implantes de Medicamentos/química , Etopósido/administración & dosificación , Etopósido/uso terapéutico , Neuroblastoma/tratamiento farmacológico , Seda/química , Animales , Antineoplásicos Fitogénicos/farmacocinética , Antineoplásicos Fitogénicos/farmacología , Bombyx/química , Línea Celular Tumoral , Preparaciones de Acción Retardada/química , Sistemas de Liberación de Medicamentos , Etopósido/farmacocinética , Etopósido/farmacología , Femenino , Humanos , Ratones Desnudos , Neuroblastoma/patologíaRESUMEN
Neuroblastomas with a high mitosis-karyorrhexis index (High-MKI) are often associated with MYCN amplification, MYCN protein overexpression and adverse clinical outcome. However, the prognostic effect of MYC-family protein expression on these neuroblastomas is less understood, especially when MYCN is not amplified. To address this, MYCN and MYC protein expression in High-MKI cases (120 MYCN amplified and 121 non-MYCN amplified) was examined by immunohistochemistry. The majority (101) of MYCN-amplified High-MKI tumors were MYCN(+), leaving one MYC(+), 2 both(+), and 16 both(-)/(+/-), whereas non-MYCN-amplified cases appeared heterogeneous, including 7 MYCN(+), 36 MYC(+), 3 both(+), and 75 both(-)/(+/-) tumors. These MYC-family proteins(+), or MYC-family driven tumors, were most likely to have prominent nucleolar (PN) formation (indicative of augmented rRNA synthesis). High-MKI neuroblastoma patients showed a poor survival irrespective of MYCN amplification. However, patients with MYC-family driven High-MKI neuroblastomas had significantly lower survival than those with non-MYC-family driven tumors. MYCN(+), MYC-family protein(+), PN(+), and clinical stage independently predicted poor survival. Specific inhibition of hyperactive rRNA synthesis and protein translation was shown to be an effective way to suppress MYC/MYCN protein expression and neuroblastoma growth. Together, MYC-family protein overexpression and PN formation should be included in new neuroblastoma risk stratification and considered for potential therapeutic targets.
RESUMEN
Background Although MYCN (aka N-myc) amplification is reported in â¼20% of neuroblastomas, MYC (aka C-myc) amplification appears to be a rare event in this disease. As of today, only 2 MYC-amplified neuroblastomas have been briefly mentioned in the literature. Methods We studied here the clinicopathological features of 3 MYC-amplified neuroblastomas. Results All 3 patients (2 females and 1 male) had stage 4 disease. One female is currently alive and well 52 months after the diagnosis, while the other female and male patients died of disease 24 and 20 months after the diagnosis, respectively. Further analysis on 2 tumors revealed unfavorable histology with MYC protein overexpression but with neither MYCN amplification nor MYCN protein overexpression. Both of these tumors exhibited "large cell neuroblastoma" histology with enlarged, uniquely open nuclei and nucleolar hypertrophy, along with "aberrant" desmin expression. Conclusions MYC-amplified neuroblastomas are extremely rare and seem to present with distinct clinicopathological features.
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Neoplasias de la Corteza Suprarrenal/patología , Amplificación de Genes/fisiología , Neuroblastoma/patología , Proteínas Proto-Oncogénicas c-myc/genética , Neoplasias de la Corteza Suprarrenal/genética , Preescolar , Femenino , Humanos , Masculino , Neuroblastoma/genéticaRESUMEN
Histone deacetylase 8 (HDAC8) is a promising drug target for multiple therapeutic applications. Here, we describe the modeling, design, synthesis, and biological evaluation of a novel series of C1-substituted tetrahydroisoquinoline (TIQ)-based HDAC8 inhibitors. Minimization of entropic loss upon ligand binding and use of the unique HDAC8 "open" conformation of the binding site yielded a successful strategy for improvement of both HDAC8 potency and selectivity. The TIQ-based 3g and 3n exhibited the highest 82 and 55 nM HDAC8 potency and 330- and 135-fold selectivity over HDAC1, respectively. Selectivity over other class I isoforms was comparable or better, whereas inhibition of HDAC6, a class II HDAC isoform, was below 50% at 10 µM. The cytotoxicity of 3g and 3n was evaluated in neuroblastoma cell lines, and 3n displayed concentration-dependent cytotoxicity similar to or better than that of PCI-34051. The selectivity of 3g and 3n was confirmed in SH-SY5Y cells as both did not increase the acetylation of histone H3 and α-tubulin. Discovery of the novel TIQ chemotype paves the way for the development of HDAC8 selective inhibitors for therapeutic applications.