RESUMEN
Immunotherapy is revolutionizing cancer treatment; however, complete responses are achieved in only a small fraction of patients and tumor types. Thus, there is an urgent need for predictive preclinical models to drive rational immunotherapeutic drug development, treatment combinations, and to minimize failures in clinical trials. Humanized mouse models (HIS) have been developed to study and modulate the interactions between immune components and tumors of human origin. In this review, we discuss recent advances in the 'humanization' of mouse models to improve the quality of human immune cell reconstitution. We also highlight new insights into the basic mechanisms, and provide a preclinical evaluation of onco-immunotherapies, as well as the limitations thereof, which constitute drivers for the improvement of the models to increase their translational power.
Asunto(s)
Neoplasias/inmunología , Escape del Tumor/inmunología , Microambiente Tumoral/inmunología , Animales , Modelos Animales de Enfermedad , Humanos , Sistema Inmunológico/citología , Sistema Inmunológico/inmunología , Sistema Inmunológico/metabolismo , Huésped Inmunocomprometido/genética , Huésped Inmunocomprometido/inmunología , Inmunoterapia/métodos , Ratones , Ratones Transgénicos , Neoplasias/genética , Neoplasias/patología , Neoplasias/terapia , Investigación , Escape del Tumor/genética , Microambiente Tumoral/genéticaRESUMEN
Nanodiamonds (NDs) are emerging delivery systems with biomedical applications and interesting perspectives in oncology. Their use has been proposed to assist the internalization of anticancer drugs and to decrease administered drug doses. The pro-apoptotic peptide ERα17p, which is issued from the hinge/N-terminus parts of the AF2 region of the human estrogen receptor α (ERα), is active at a concentration of 10µM on breast cancer cells and particularly on those cancer cells that are ERα-positive. We have synthesized ND@ERα17p conjugates by physisorption of the cationic peptide ERα17p on the surface of anionic NDs. Resulting ND@ERα17p suspensions were characterized by far-UV electronic circular dichroism (ECD), dynamic light scattering (DLS) and zetametry. We then tested the anti-proliferative action of ND@ERα17p on ERα-positive MCF-7 breast carcinoma cells. ND@ERα17p allowed a decrease of the active concentration to 0.1nM (ND@ERα17p), revealing unambiguously that NDs could be used to improve the anti-proliferative action of this peptide. This preliminary study proposes a novel approach for enhancing the apoptotic action displayed by ERα17p, in the context of breast cancer.
Asunto(s)
Adenocarcinoma/patología , Neoplasias de la Mama/patología , Nanoconjugados , Nanodiamantes , Fragmentos de Péptidos/farmacología , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , Dicroismo Circular , Portadores de Fármacos , Diseño de Fármacos , Dispersión Dinámica de Luz , Receptor alfa de Estrógeno/química , Femenino , Humanos , Células MCF-7 , Microscopía Electrónica , Fragmentos de Péptidos/química , Electricidad EstáticaRESUMEN
BACKGROUND: Oestrogen receptor-negative (ER-) breast cancer is intrinsically sensitive to chemotherapy. However, tumour response is often incomplete, and relapse occurs with high frequency. The aim of this work was to analyse the molecular characteristics of residual tumours and early response to chemotherapy in patient-derived xenografts (PDXs) of breast cancer. METHODS: Gene and protein expression profiles were analysed in a panel of ER- breast cancer PDXs before and after chemotherapy treatment. Tumour and stromal interferon-gamma expression was measured in xenografts lysates by human and mouse cytokine arrays, respectively. RESULTS: The analysis of residual tumour cells in chemo-responder PDX revealed a strong overexpression of IFN-inducible genes, induced early after AC treatment and associated with increased STAT1 phosphorylation, DNA-damage and apoptosis. No increase in IFN-inducible gene expression was observed in chemo-resistant PDXs upon chemotherapy. Overexpression of IFN-related genes was associated with human IFN-γ secretion by tumour cells. CONCLUSIONS: Treatment-induced activation of the IFN/STAT1 pathway in tumour cells is associated with chemotherapy response in ER- breast cancer. Further validations in prospective clinical trials will aim to evaluate the usefulness of this signature to assist therapeutic strategies in the clinical setting.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Interferón gamma/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Factor de Transcripción STAT1/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales , Animales , Antígenos/efectos de los fármacos , Antígenos/genética , Antígenos/metabolismo , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Capecitabina/farmacología , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Caspasa 3/efectos de los fármacos , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 7/efectos de los fármacos , Caspasa 7/genética , Caspasa 7/metabolismo , Cisplatino/farmacología , Citocinas/efectos de los fármacos , Citocinas/genética , Citocinas/metabolismo , Proteínas del Citoesqueleto/efectos de los fármacos , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Hibridación in Situ , Interferón beta/efectos de los fármacos , Interferón beta/genética , Interferón beta/metabolismo , Interferón gamma/genética , Interferón gamma/metabolismo , Péptidos y Proteínas de Señalización Intracelular/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Desnudos , Proteínas Mitocondriales/efectos de los fármacos , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas de Resistencia a Mixovirus/efectos de los fármacos , Proteínas de Resistencia a Mixovirus/genética , Proteínas de Resistencia a Mixovirus/metabolismo , Trasplante de NeoplasiasRESUMEN
BACKGROUND: In experimental models, bevacizumab suppressed in vitro growth and in vivo hepatic metastasis of ocular melanoma cells. Additional preclinical data suggested a potential benefit when combining bevacizumab with dacarbazine. METHODS: This noncomparative phase II study evaluated a combination of bevacizumab (10 mg/kg on days 8 and 22) with temozolomide (150 mg/m(2) on days 1-7 and 15-21) in 36 patients with metastatic uveal melanoma (MUM). The primary endpoint was the progression-free rate (PFR) at 6 months. Using a modified 2-step Fleming plan, at least 10 of 35 patients were required to support a predefined PFR at 6 months of 40%. Secondary objectives were progression-free survival (PFS), overall survival (OS), and safety; liver perfusion computed tomography (CT) for response imaging; and impact of VEGF-A gene polymorphisms on bevacizumab pharmacodynamics. RESULTS: First- and second-step analyses revealed nonprogression at 6 months in 3 of 17 and 8 of 35 patients, respectively. Finally, the 6-month PFR was 23% (95% confidence interval [CI]: 10-39), with long-lasting stable disease in 5 patients (14%). Median PFS and OS were 12 weeks and 10 months, respectively. No unexpected toxicity occurred. Liver perfusion CT imaging was not useful in assessing tumor response, and VEGF-A gene polymorphisms were not correlated with toxicity or survival. CONCLUSION: In patients with MUM, a combination of bevacizumab plus temozolomide achieved a 6-month PFR of 23%.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Bevacizumab/uso terapéutico , Dacarbazina/análogos & derivados , Melanoma/tratamiento farmacológico , Melanoma/genética , Neoplasias de la Úvea/tratamiento farmacológico , Neoplasias de la Úvea/genética , Factor A de Crecimiento Endotelial Vascular/genética , Adulto , Anciano , Bevacizumab/efectos adversos , Dacarbazina/efectos adversos , Dacarbazina/uso terapéutico , Supervivencia sin Enfermedad , Esquema de Medicación , Femenino , Humanos , Masculino , Melanoma/patología , Persona de Mediana Edad , Imagen de Perfusión , Temozolomida , Tomografía Computarizada por Rayos X , Resultado del Tratamiento , Neoplasias de la Úvea/patologíaRESUMEN
Several publications have suggested that histone deacetylase inhibitors (HDACis) could reverse the repression of estrogen receptor alpha (ERα) in triple-negative breast cancer (TNBC) cell lines, leading to the induction of a functional protein. Using different HDACis, vorinostat, panobinostat, and abexinostat, we therefore investigated this hypothesis in various human TNBC cell lines and patient-derived xenografts (PDXs). We used three human TNBC cell lines and three PDXs. We analyzed the in vitro toxicity of the compounds, their effects on the hormone receptors and hormone-related genes and protein expression both in vitro and in vivo models. We then explored intra-tumor histone H3 acetylation under abexinostat in xenograft models. Despite major cytotoxicity of all tested HDAC inhibitors and repression of deactylation-dependent CCND1 gene, neither ERα nor ERß, ESR1 or ESR2 genes respectively, were re-expressed in vitro. In vivo, after administration of abexinostat for three consecutive days, we did not observe any induction of ESR1 or ESR1-related genes and ERα protein expression by RT-qPCR and immunohistochemical methods in PDXs. This observation was concomitant to the fact that in vivo administration of abexinostat increased intra-tumor histone H3 acetylation. These observations do not allow us to confirm previous studies which suggested that HDACis are able to convert ER-negative (ER-) tumors to ER-positive (ER+) tumors, and that a combination of HDAC inhibitors and hormone therapy could be proposed in the management of TNBC patients.
Asunto(s)
Ciclina D1/biosíntesis , Receptor alfa de Estrógeno/genética , Receptor beta de Estrógeno/biosíntesis , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Benzofuranos/administración & dosificación , Proliferación Celular/efectos de los fármacos , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor beta de Estrógeno/antagonistas & inhibidores , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/administración & dosificación , Histonas/genética , Humanos , Ácidos Hidroxámicos/administración & dosificación , Indoles/administración & dosificación , Panobinostat , Receptor ErbB-2/genética , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Vorinostat , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The B-subunit of Shiga toxin (STxB) specifically binds to the glycosphingolipid Gb3 that is highly expressed on a number of human tumors and has been shown to target tumor cells in mouse models and ex vivo on primary colon carcinoma specimen. METHODS: Using a novel ex vivo STxB labeling (ESL) method we studied Gb3 expression in cytological specimens of primary human breast tumors from 107 patients, and in synchronous lymph node metastases from 20 patients. Fluorescent STxB was incubated with fine-needle aspiration (FNA) specimens, and Gb3 expression was evaluated by fluorescence microscopy. Furthermore, 11 patient-derived human breast cancer xenografts (HBCx) were evaluated for expression of Gb3 by ESL and FACS. In addition, the biodistribution of fluorescent STxB conjugate was studied after intravenous injection in a Gb3 positive HBCx model. RESULTS: Gb3 expression was detected in 62 of 107 patients (57.9%), mainly in epithelial tumor cells. Gb3 positivity correlated with estrogen receptor expression (p≤0.01), whereas absence of Gb3 expression in primary tumors was correlated with the presence of lymph node metastases (p≤0.03). 65% of lymph node metastases were Gb3 positive and in 40% of tested patients, we observed a statistically significant increase of metastatic Gb3 expression (p≤0.04). Using concordant ESL and flow cytometry analysis, 6 out of 11 HBCx samples were scored positive. Intravenous injections of fluorescent STxB into HBC xenografted mice showed preferential STxB accumulation in epithelial cells and cells with endothelial morphology of the tumor. CONCLUSION: The enhanced expression of Gb3 in primary breast carcinomas and its lymph node metastases indicate that the development of STxB-based therapeutic strategies is of interest in this pathology. Gb3 expressing HBCx can be used as a model for preclinical studies with STxB conjugates. Finally, the ESL technique on FNA represents a rapid and cost effective method for the stratification of patients in future clinical trials.
Asunto(s)
Adenofibroma/química , Antígenos de Carbohidratos Asociados a Tumores/análisis , Neoplasias de la Mama/química , Carcinoma/química , Toxinas Shiga/farmacocinética , Animales , Biopsia con Aguja Fina , Mama/química , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma/metabolismo , Carcinoma/secundario , Sistemas de Liberación de Medicamentos , Femenino , Citometría de Flujo , Humanos , Inyecciones Intravenosas , Metástasis Linfática , Glándulas Mamarias Humanas/química , Ratones , Microscopía Fluorescente , Persona de Mediana Edad , Receptores de Estrógenos/análisis , Toxinas Shiga/administración & dosificaciónRESUMEN
BACKGROUND: Tumor endothelial transdifferentiation and VEGFR1/2 expression by cancer cells have been reported in glioblastoma but remain poorly documented for many other cancer types. METHODS: To characterize vasculature of patient-derived tumor xenografts (PDXs), largely used in preclinical anti-angiogenic assays, we designed here species-specific real-time quantitative RT-PCR assays. Human and mouse PECAM1/CD31, ENG/CD105, FLT1/VEGFR1, KDR/VEGFR2 and VEGFA transcripts were analyzed in a large series of 150 PDXs established from 8 different tumor types (53 colorectal, 14 ovarian, 39 breast and 15 renal cell cancers, 6 small cell and 5 non small cell lung carcinomas, 13 cutaneous melanomas and 5 glioblastomas) and in two bevacizumab-treated non small cell lung carcinomas xenografts. RESULTS: As expected, mouse cell proportion in PDXs -evaluated by quantifying expression of the housekeeping gene TBP- correlated with all mouse endothelial markers and human VEGFA RNA levels. More interestingly, we observed human PECAM1/CD31 and ENG/CD105 expression in all tumor types, with higher rate in glioblastoma and renal cancer xenografts. Human VEGFR expression profile varied widely depending on tumor types with particularly high levels of human FLT1/VEGFR1 transcripts in colon cancers and non small cell lung carcinomas, and upper levels of human KDR/VEGFR2 transcripts in non small cell lung carcinomas. Bevacizumab treatment induced significant low expression of mouse Pecam1/Cd31, Eng/Cd105, Flt1/Vegfr1 and Kdr/Vefr2 while the human PECAM1/CD31 and VEGFA were upregulated. CONCLUSIONS: Taken together, our results strongly suggest existence of human tumor endothelial cells in all tumor types tested and of both stromal and tumoral autocrine VEGFA-VEGFR1/2 signalings. These findings should be considered when evaluating molecular mechanisms of preclinical response and resistance to tumor anti-angiogenic strategies.
Asunto(s)
Células Endoteliales/metabolismo , Perfilación de la Expresión Génica/métodos , Neoplasias Experimentales/genética , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/genética , Inhibidores de la Angiogénesis/farmacología , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Bevacizumab , Biomarcadores de Tumor/metabolismo , Células Endoteliales/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Calcineurin is a calcium-activated serine/threonine phosphatase critical to a number of developmental processes in the cardiovascular, nervous and immune systems. In the T-cell lineage, calcineurin activation is important for pre-T-cell receptor (TCR) signaling, TCR-mediated positive selection of thymocytes into mature T cells, and many aspects of the immune response. The critical role of calcineurin in the immune response is underscored by the fact that calcineurin inhibitors, such as cyclosporin A (CsA) and FK506, are powerful immunosuppressants in wide clinical use. We observed sustained calcineurin activation in human B- and T-cell lymphomas and in all mouse models of lymphoid malignancies analyzed. In intracellular NOTCH1 (ICN1)- and TEL-JAK2-induced T-cell lymphoblastic leukemia, two mouse models relevant to human malignancies, in vivo inhibition of calcineurin activity by CsA or FK506 induced apoptosis of leukemic cells and rapid tumor clearance, and substantially prolonged mouse survival. In contrast, ectopic expression of a constitutively activated mutant of calcineurin favored leukemia progression. Moreover, CsA treatment induced apoptosis in human lymphoma and leukemia cell lines. Thus, calcineurin activation is critical for the maintenance of the leukemic phenotype in vivo, identifying this pathway as a relevant therapeutic target in lymphoid malignancies.
Asunto(s)
Antineoplásicos/farmacología , Calcineurina/metabolismo , Leucemia-Linfoma de Células T del Adulto/tratamiento farmacológico , Leucemia-Linfoma de Células T del Adulto/enzimología , Animales , Inhibidores de la Calcineurina , Línea Celular Tumoral , Ciclosporina/farmacología , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Humanos , Leucemia-Linfoma de Células T del Adulto/patología , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/enzimología , Linfoma de Células B/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas de Fusión Oncogénica/deficiencia , Proteínas de Fusión Oncogénica/genética , Receptor Notch1/fisiología , Tacrolimus/farmacologíaRESUMEN
Metabolic adaptations and changes in the expression of nutrient transporters are known to accompany tumorigenic processes. Nevertheless, in the context of solid tumors, studies of metabolism are hindered by a paucity of tools allowing the identification of cell surface transporters on individual cells. Here, we developed a method for the dissociation of human breast cancer tumor xenografts combined with quantification of cell surface markers, including metabolite transporters. The expression profiles of four relevant nutrient transporters for cancer cells' metabolism, Glut1, ASCT2, PiT1 and PiT2 (participating to glucose, glutamine and inorganic phosphate, respectively), as detected by new retroviral envelope glycoprotein-derived ligands, were distinctive of each tumor, unveiling underlying differences in metabolic pathways. Our tumor dissociation procedure and nutrient transporter profiling technology provides opportunities for future basic research, clinical diagnosis, prognosis and evaluation of therapeutic responses, as well as for drug discovery and development.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Citometría de Flujo/métodos , Proteínas de Transporte de Membrana/metabolismo , Análisis de Varianza , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Supervivencia Celular/fisiología , Femenino , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Inmunohistoquímica/métodos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Reproducibilidad de los Resultados , Trasplante HeterólogoRESUMEN
This international, multicentre phase II study was conducted to assess ofatumumab, a human anti-CD20 monoclonal antibody, in patients with relapsed/progressive diffuse large B-cell lymphoma (DLBCL) who were ineligible for autologous stem cell transplantation (TI) or who had relapse/progression after transplantation (PT). Eighty-one patients received ofatumumab 300 mg intravenously (IV) on Day 1, followed by seven weekly IV infusions of 1000 mg. Patients in the TI and PT groups had received a median of 3 (range, 1-7) and 5 (range, 2-7) prior therapies, respectively. One-third of patients did not respond to the last prior therapy, and 53% had failed two or more rituximab-containing therapies. Overall response rate was 13% for the TI group (seven partial responses) and 8% for the PT group (two complete responses). Median progression-free survival was 2·6 months, and median duration of response was 9·5 months. The most common Grade 3-4 adverse events were neutropenia (11%), leucopenia (6%), lymphopenia (6%) and thrombocytopenia (6%). Sixteen deaths have been reported, with disease progression as the most common cause of death. In conclusion, ofatumumab monotherapy was well tolerated and provided clinical benefit to some DLBCL patients in this study.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Inmunoterapia , Linfoma de Células B Grandes Difuso/terapia , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales Humanizados , Anticuerpos Monoclonales de Origen Murino/administración & dosificación , Antígenos CD20/inmunología , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Antineoplásicos Alquilantes/administración & dosificación , Antineoplásicos Alquilantes/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Terapia Combinada , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos , Femenino , Enfermedades Hematológicas/inducido químicamente , Trasplante de Células Madre Hematopoyéticas , Humanos , Inmunoterapia/efectos adversos , Estimación de Kaplan-Meier , Linfoma de Células B Grandes Difuso/cirugía , Masculino , Persona de Mediana Edad , Recurrencia , Inducción de Remisión , Rituximab , Terapia Recuperativa , Adulto JovenRESUMEN
Long-term responses have been reported after autologous stem cell transplantation (ASCT) for chronic lymphocytic leukemia (CLL). We conducted a prospective, randomized trial of ASCT in previously untreated CLL patients. We enrolled 241 patients < 66 years of age with Binet stage B or C CLL. They received 3 courses of mini-CHOP (cyclophosphamide, hydroxydaunorubicin, oncovin, and prednisone/prednisolone) and then 3 courses of fludarabine. Patients in complete response (CR) were then randomized to ASCT or observation, whereas the other patients were randomized to dexamethasone, high-dose aracytin, cisplatin (DHAP) salvage followed by either ASCT or 3 courses of fludarabine plus cyclophosphamide (FC). The primary end point was event-free survival (EFS). After up-front treatment, 105 patients entered CR and were randomized between ASCT (n = 52) and observation (n = 53); their respective 3-year EFS rates were 79.8% and 35.5%; the adjusted hazard ratio was 0.3 (95% CI: 0.1-0.7; P = .003). Ninety-four patients who did not enter CR were randomized between ASCT (n = 46) and FC (n = 48); their respective 3-year EFS rates were 48.9% and 44.4%, respectively; the adjusted hazard ratio was 1.7 (95% CI: 0.9-3.2; P = .13). No difference in overall survival was found between the 2 response subgroups. In young CLL patients in CR, ASCT consolidation markedly delayed disease progression. No difference was observed between ASCT and FC in patients requiring DHAP salvage.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Leucemia Linfocítica Crónica de Células B/mortalidad , Leucemia Linfocítica Crónica de Células B/terapia , Trasplante de Células Madre , Adolescente , Adulto , Factores de Edad , Anciano , Cisplatino/administración & dosificación , Ciclofosfamida/administración & dosificación , Citarabina/administración & dosificación , Dexametasona/administración & dosificación , Supervivencia sin Enfermedad , Doxorrubicina/administración & dosificación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Prednisona/administración & dosificación , Tasa de Supervivencia , Factores de Tiempo , Trasplante Autólogo , Vincristina/administración & dosificaciónRESUMEN
The in vitro to in vivo translation of metal-based cytotoxic drugs has proven to be a significant hurdle in their establishment as effective anti-cancer alternatives. Various nano-delivery systems, such as polymeric nanoparticles, have been explored to address the pharmacokinetic limitations associated with the use of these complexes. However, these systems often suffer from poor stability or involve complex synthetic procedures. To circumvent these problems, we report here a simple, one-pot procedure for the preparation of covalently-attached Ru-polylactide nanoparticles. This methodology relies on the ring-opening polymerization of lactide initiated by a calcium alkoxide derivative formed from calcium bis(trimethylsilyl amide) and a hydroxyl-bearing ruthenium complex. This procedure proceeds with high efficiency (near-quantitative incorporation of Ru in the polymer) and enables the preparation of polymers with varying molecular weights (2000-11000 Da) and high drug loadings (up to 68% w/w). These polymers were formulated as narrowly dispersed nanoparticles (110 nm) that exhibited a slow and predictable release of the ruthenium payload. Unlike standard encapsulation methods routinely used, the release kinetics of these nanoparticles is controlled and may be adjusted on demand, by tuning the size of the polymer chain. In terms of cytotoxicity, the nanoparticles were assessed in the ovarian cancer cell line A2780 and displayed potency comparable to cisplatin and the free drug, in the low micromolar range. Interestingly, the activity was maintained when tested in a cisplatin-resistant cell line, suggesting a possible orthogonal mechanism of action. Additionally, the internalization in tumour cells was found to be significantly higher than the free ruthenium complex (>200 times in some cases), clearly showcasing the added benefit in the drug's cellular permeation and accumulation of the drug. Finally, the in vivo performance was evaluated for the first time in mice. The experiments showed that the intravenously injected nanoparticles were well tolerated and were able to significantly improve the pharmacokinetics and biodistribution of the parent drug. Not only was the nanosystem able to promote an 18-fold increase in tumour accumulation, but it also allowed a considerable reduction of drug accumulation in vital organs, achieving, for example, reduction levels of 90% and 97% in the brain and lungs respectively. In summary, this simple and efficient one-pot procedure enables the generation of stable and predictable nanoparticles capable of improving the cellular penetration and systemic accumulation of the Ru drug in the tumour. Altogether, these results showcase the potential of covalently-loaded ruthenium polylactide nanoparticles and pave the way for its exploitation and application as a viable tool in the treatment of ovarian cancer.
RESUMEN
The synthetic peptide ERα17p (sequence: PLMIKRSKKNSLALSLT), which corresponds to the 295-311 region of the human estrogen receptor α (ERα), induces apoptosis in breast cancer cells. In mice and at low doses, it promotes not only the decrease of the size of xenografted triple-negative human breast tumors, but also anti-inflammatory and anti-nociceptive effects. Recently, we have shown that these effects were due to its interaction with the seven-transmembrane G protein-coupled estrogen receptor GPER. Following modeling studies, the C-terminus of this peptide (sequence: NSLALSLT) remains compacted at the entrance of the GPER ligand-binding pocket, whereas its N-terminus (sequence: PLMI) engulfs in the depth of the same pocket. Thus, we have hypothesized that the PLMI motif could support the pharmacological actions of ERα17p. Here, we show that the PLMI peptide is, indeed, responsible for the GPER-dependent antiproliferative and anti-nociceptive effects of ERα17p. By using different biophysical approaches, we demonstrate that the NSLALSLT part of ERα17p is responsible for aggregation. Overall, the tetrapeptide PLMI, which supports the action of the parent peptide ERα17p, should be considered as a hit for the synthesis of new GPER modulators with dual antiproliferative and anti-nociceptive actions. This study highlights also the interest to modulate GPER for the control of pain.
Asunto(s)
Receptor alfa de Estrógeno , Neoplasias de la Mama Triple Negativas , Animales , Humanos , Ratones , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Estrógenos , Péptidos , Receptores Acoplados a Proteínas G , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Uveal melanoma (UM) has a high risk to progress to metastatic disease with a median survival of 3.9 months after metastases detection, as metastatic UM responds poorly to conventional and targeted chemotherapy and is largely refractory to immunotherapy. Here, we present a patient-derived zebrafish UM xenograft model mimicking metastatic UM. Cells isolated from Xmm66 spheroids derived from metastatic UM patient material were injected into 2 days-old zebrafish larvae resulting in micro-metastases in the liver and caudal hematopoietic tissue. Metastasis formation could be reduced by navitoclax and more efficiently by the combinations navitoclax/everolimus and flavopiridol/quisinostat. We obtained spheroid cultures from 14 metastatic and 10 primary UM tissues, which were used for xenografts with a success rate of 100%. Importantly, the ferroptosis-related genes GPX4 and SLC7A11 are negatively correlated with the survival of UM patients (TCGA: n = 80; Leiden University Medical Centre cohort: n = 64), ferroptosis susceptibility is correlated with loss of BAP1, one of the key prognosticators for metastatic UM, and ferroptosis induction greatly reduced metastasis formation in the UM xenograft model. Collectively, we have established a patient-derived animal model for metastatic UM and identified ferroptosis induction as a possible therapeutic strategy for the treatment of UM patients.
RESUMEN
Uveal Melanoma (UM) is a rare and malignant intraocular tumor with dismal prognosis. Even if radiation or surgery permit an efficient control of the primary tumor, up to 50% of patients subsequently develop metastases, mainly in the liver. The treatment of UM metastases is challenging and the patient survival is very poor. The most recurrent event in UM is the activation of Gαq signaling induced by mutations in GNAQ/11. These mutations activate downstream effectors including protein kinase C (PKC) and mitogen-activated protein kinases (MAPK). Clinical trials with inhibitors of these targets have not demonstrated a survival benefit for patients with UM metastasis. Recently, it has been shown that GNAQ promotes YAP activation through the focal adhesion kinase (FAK). Pharmacological inhibition of MEK and FAK showed remarkable synergistic growth-inhibitory effects in UM both in vitro and in vivo. In this study, we have evaluated the synergy of the FAK inhibitor with a series of inhibitors targeting recognized UM deregulated pathways in a panel of cell lines. The combined inhibition of FAK and MEK or PKC had highly synergistic effects by reducing cell viability and inducing apoptosis. Furthermore, we demonstrated that these combinations exert a remarkable in vivo activity in UM patient-derived xenografts. Our study confirms the previously described synergy of the dual inhibition of FAK and MEK and identifies a novel combination of drugs (FAK and PKC inhibitors) as a promising strategy for therapeutic intervention in metastatic UM.
RESUMEN
Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited. Here, we explored the use of xenografts in zebrafish for high-throughput drug screening to discover new combination therapies for Ewing sarcoma. We subjected xenografts in zebrafish larvae to high-content imaging and subsequent automated tumor size analysis to screen single agents and compound combinations. We identified three drug combinations effective against Ewing sarcoma cells: Irinotecan combined with either an MCL-1 or an BCL-XL inhibitor and in particular dual inhibition of the anti-apoptotic proteins MCL-1 and BCL-XL, which efficiently eradicated tumor cells in zebrafish xenografts. We confirmed enhanced efficacy of dual MCL-1/BCL-XL inhibition compared to single agents in a mouse PDX model. In conclusion, high-content screening of small compounds on Ewing sarcoma zebrafish xenografts identified dual MCL-1/BCL-XL targeting as a specific vulnerability and promising therapeutic strategy for Ewing sarcoma, which warrants further investigation towards clinical application.
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Sarcoma de Ewing , Humanos , Animales , Ratones , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Pez Cebra/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Evaluación Preclínica de Medicamentos , Xenoinjertos , Apoptosis , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Línea Celular TumoralRESUMEN
Uveal melanoma is the most common primary intraocular malignancy in adults. Up to 50% of UM patients develop metastatic disease, usually in the liver. When metastatic, the prognosis is poor, and few treatment options exist. Here, we investigated the feasibility of establishing patient-derived xenografts (PDXs) from a patient's tumor in order to screen for therapies that the patient could benefit from. Samples obtained from 29 primary tumors and liver metastases of uveal melanoma were grafted into SCID mice. PDX models were successfully established for 35% of primary patient tumors and 67% of liver metastases. The tumor take rate was proportional to the risk of metastases. PDXs showed the same morphology, the same GNAQ/11, BAP1, and SF3B1 mutations, and the same chromosome 3 and 8q status as the corresponding patient samples. Six PDX models were challenged with two compounds for 4 weeks. We show that, for 31% of patients with high or intermediate risk of metastasis, the timing to obtain efficacy results on PDX models derived from their primary tumors was compatible with the selection of the therapy to treat the patient after relapse. PDXs could thus be a valid tool ("avatar") to select the best personalized therapy for one third of patients that are most at risk of relapse.
Asunto(s)
Neoplasias Hepáticas , Recurrencia Local de Neoplasia , Adulto , Animales , Ratones , Humanos , Estudios de Factibilidad , Xenoinjertos , Ratones SCID , Neoplasias Hepáticas/genética , RecurrenciaRESUMEN
OBJECTIVE: Chordomas are rare bone neoplasms characterized by a high recurrence rate and no benefit from any approved medical treatment to date. However, the investigation of molecular alterations in chordomas could be essential to prognosticate, guide clinical decision-making, and identify theranostic biomarkers. The aim of this study was to provide a detailed genomic landscape of a homogeneous series of 64 chordoma samples, revealing driver events, theranostic markers, and outcome-related genomic features. METHODS: The authors conducted whole-exome sequencing (WES), targeted next-generation sequencing, and RNA sequencing of 64 skull base and spinal chordoma samples collected between December 2006 and September 2020. Clinical, histological, and radiological data were retrospectively analyzed and correlated to genetic findings. RESULTS: The authors identified homozygous deletions of CDKN2A/2B, PIK3CA mutations, and alterations affecting genes of SWI/SNF chromatin remodeling complexes (PBRM1 and ARID1A) as potential theranostic biomarkers. Using matched germline WES, they observed a higher frequency of a common genetic variant (rs2305089; p.(Gly177Asp)) in TBXT (97.8%, p < 0.001) compared to its distribution in the general population. PIK3CA mutation was identified as an independent biomarker of short progression-free survival (HR 10.68, p = 0.0008). Loss of CDKN2A/2B was more frequently observed in spinal tumors and recurrent tumors. CONCLUSIONS: In the current study, the authors identified driver events such as PBRM1 and PIK3CA mutations, TBXT alterations, or homozygous deletions of CDKN2A/2B, which could, for some, be considered potential theranostic markers and could allow for identifying novel therapeutic approaches. With the aim of a future biomolecular prognostication classification, alterations affecting PIK3CA and CDKN2A/2B could be considered as poor prognostic biomarkers.
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Cordoma , Neoplasias de la Base del Cráneo , Neoplasias de la Columna Vertebral , Humanos , Pronóstico , Cordoma/patología , Neoplasias de la Columna Vertebral/genética , Medicina de Precisión , Estudios Retrospectivos , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Biomarcadores , Neoplasias de la Base del Cráneo/patología , Base del Cráneo/patología , Fosfatidilinositol 3-Quinasa Clase I/genéticaRESUMEN
PURPOSE: ALK-activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1%-2% of cases. Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK) inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single-agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data have suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway. EXPERIMENTAL DESIGN: We compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin, and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient-derived xenografts (PDX). RESULTS: Lorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK-amplified PDX model with high ALK expression. In this PDX, lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth. CONCLUSIONS: In our preclinical neuroblastoma models, high ALK expression was associated with lorlatinib response alone or in combination with either chemotherapy or idasanutlin. The synergy between MDM2 and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma.
Asunto(s)
Neoplasias Pulmonares , Neuroblastoma , Ratones , Animales , Humanos , Quinasa de Linfoma Anaplásico/genética , Aminopiridinas/uso terapéutico , Lactamas Macrocíclicas/farmacología , Lactamas Macrocíclicas/uso terapéutico , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológicoRESUMEN
Uveal melanoma (UM) is a rare cancer resulting from the transformation of melanocytes in the uveal tract. Integrative analysis has identified four molecular and clinical subsets of UM. To improve our molecular understanding of UM, we performed extensive multi-omics characterization comparing two aggressive UM patient-derived xenograft models with normal choroidal melanocytes, including DNA optical mapping, specific histone modifications, and DNA topology analysis using Hi-C. Our gene expression and cytogenetic analyses suggest that genomic instability is a hallmark of UM. We also identified a recurrent deletion in the BAP1 promoter resulting in loss of expression and associated with high risk of metastases in UM patients. Hi-C revealed chromatin topology changes associated with the upregulation of PRAME, an independent prognostic biomarker in UM, and a potential therapeutic target. Our findings illustrate how multi-omics approaches can improve our understanding of tumorigenesis and reveal two distinct mechanisms of gene expression dysregulation in UM.