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1.
Cell ; 175(4): 1014-1030.e19, 2018 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-30343900

RESUMEN

Although current immune-checkpoint therapy (ICT) mainly targets lymphoid cells, it is associated with a broader remodeling of the tumor micro-environment. Here, using complementary forms of high-dimensional profiling, we define differences across all hematopoietic cells from syngeneic mouse tumors during unrestrained tumor growth or effective ICT. Unbiased assessment of gene expression of tumor-infiltrating cells by single-cell RNA sequencing (scRNAseq) and longitudinal assessment of cellular protein expression by mass cytometry (CyTOF) revealed significant remodeling of both the lymphoid and myeloid intratumoral compartments. Surprisingly, we observed multiple subpopulations of monocytes/macrophages, distinguishable by the markers CD206, CX3CR1, CD1d, and iNOS, that change over time during ICT in a manner partially dependent on IFNγ. Our data support the hypothesis that this macrophage polarization/activation results from effects on circulatory monocytes and early macrophages entering tumors, rather than on pre-polarized mature intratumoral macrophages.


Asunto(s)
Linfocitos/inmunología , Células Mieloides/inmunología , Neoplasias/inmunología , Análisis de la Célula Individual , Transcriptoma , Animales , Línea Celular Tumoral , Citometría de Flujo , Inmunoterapia/métodos , Interferón gamma/inmunología , Activación de Macrófagos , Masculino , Espectrometría de Masas , Ratones , Células Precursoras de Monocitos y Macrófagos/inmunología , Neoplasias/terapia
3.
Nature ; 574(7780): 696-701, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31645760

RESUMEN

The ability of the immune system to eliminate and shape the immunogenicity of tumours defines the process of cancer immunoediting1. Immunotherapies such as those that target immune checkpoint molecules can be used to augment immune-mediated elimination of tumours and have resulted in durable responses in patients with cancer that did not respond to previous treatments. However, only a subset of patients benefit from immunotherapy and more knowledge about what is required for successful treatment is needed2-4. Although the role of tumour neoantigen-specific CD8+ T cells in tumour rejection is well established5-9, the roles of other subsets of T cells have received less attention. Here we show that spontaneous and immunotherapy-induced anti-tumour responses require the activity of both tumour-antigen-specific CD8+ and CD4+ T cells, even in tumours that do not express major histocompatibility complex (MHC) class II molecules. In addition, the expression of MHC class II-restricted antigens by tumour cells is required at the site of successful rejection, indicating that activation of CD4+ T cells must also occur in the tumour microenvironment. These findings suggest that MHC class II-restricted neoantigens have a key function in the anti-tumour response that is nonoverlapping with that of MHC class I-restricted neoantigens and therefore needs to be considered when identifying patients who will most benefit from immunotherapy.


Asunto(s)
Antígenos de Neoplasias/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Neoplasias Experimentales/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Inmunoterapia , Ratones , Neoplasias Experimentales/terapia
4.
Proc Natl Acad Sci U S A ; 118(24)2021 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-34099555

RESUMEN

Immunotherapies are a promising advance in cancer treatment. However, because only a subset of cancer patients benefits from these treatments it is important to find mechanisms that will broaden the responding patient population. Generally, tumors with high mutational burdens have the potential to express greater numbers of mutant neoantigens. As neoantigens can be targets of protective adaptive immunity, highly mutated tumors are more responsive to immunotherapy. Given that external beam radiation 1) is a standard-of-care cancer therapy, 2) induces expression of mutant proteins and potentially mutant neoantigens in treated cells, and 3) has been shown to synergize clinically with immune checkpoint therapy (ICT), we hypothesized that at least one mechanism of this synergy was the generation of de novo mutant neoantigen targets in irradiated cells. Herein, we use KrasG12D x p53-/- sarcoma cell lines (KP sarcomas) that we and others have shown to be nearly devoid of mutations, are poorly antigenic, are not controlled by ICT, and do not induce a protective antitumor memory response. However, following one in vitro dose of 4- or 9-Gy irradiation, KP sarcoma cells acquire mutational neoantigens and become sensitive to ICT in vivo in a T cell-dependent manner. We further demonstrate that some of the radiation-induced mutations generate cytotoxic CD8+ T cell responses, are protective in a vaccine model, and are sufficient to make the parental KP sarcoma line susceptible to ICT. These results provide a proof of concept that induction of new antigenic targets in irradiated tumor cells represents an additional mechanism explaining the clinical findings of the synergy between radiation and immunotherapy.


Asunto(s)
Antígenos de Neoplasias/inmunología , Inmunoterapia , Mutación/genética , Neoplasias/genética , Neoplasias/inmunología , Radiación , Animales , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Células Clonales , Femenino , Antígenos de Histocompatibilidad Clase II/metabolismo , Proteínas de Punto de Control Inmunitario/metabolismo , Inmunidad , Ratones , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteína p53 Supresora de Tumor/genética , Vacunación
5.
Eur J Immunol ; 49(4): 534-545, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30758056

RESUMEN

Dendritic cells (DCs) are key players in immunity and tolerance. Some DCs express c-kit, the receptor for stem cell factor (SCF), nevertheless c-kit functional role and the regulation of its expression in DCs are incompletely defined. We recently demonstrated that autocrine SCF sustains a pro-survival circuit, and that SCF increases phospho-AKT in c-kit+ mouse bone marrow-derived DCs (BMdDCs). Herein we observed that CpG and PolyI:C, two stimuli mimicking bacterial and viral nucleic acids respectively, strongly inhibited c-kit expression by BMdDCs and spleen DCs in vitro and in vivo. Experiments in IFNARI-/- mice showed that IFN-I pathway was required for c-kit down-regulation in cDC1s, but only partially supported it in cDC2s. Furthermore, CpG and PolyI:C strongly inhibited c-kit mRNA expression. In agreement with the reduced c-kit levels, SCF pro-survival activity was impaired. Thus in the presence of exogenously provided SCF, either PolyI:C or CpG induced spleen DC death in 2 days, while at earlier times IL-6 and IL-12 production were slightly increased. In contrast, SCF improved survival of unstimulated spleen DCs expressing high c-kit levels. Our studies suggest that c-kit down-modulation is a previously neglected component of DC response to CpG and PolyI:C, regulating DC survival and ultimately tuning immune response.


Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Expresión Génica , Proteínas Proto-Oncogénicas c-kit/genética , Animales , Antígenos CD40/metabolismo , Células Cultivadas , Citocinas/biosíntesis , Inmunofenotipificación , Interleucina-6/biosíntesis , Ratones , Oligodesoxirribonucleótidos/inmunología , Poli I-C/inmunología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Bazo
6.
NMR Biomed ; 28(3): 317-26, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25581615

RESUMEN

Patients suffering from glioblastoma multiforme (GBM) face a poor prognosis with median survival of about 14 months. High recurrence rate and failure of conventional treatments are attributed to the presence of GBM cells with stem-like properties (GSCs). Metabolite profiles of 42 GSC lines established from the tumor tissue of adult GBM patients were screened with (1) H NMR spectroscopy and compared with human neural progenitor cells from human adult olfactory bulb (OB-NPCs) and from the developing human brain (HNPCs). A first subset (n=12) of GSCs exhibited a dramatic accumulation of the metabolite α-aminoadipate (αAAD), product of the oxidation of α-aminoadipic semialdehyde catalyzed by the ALDH7A1 aldehyde dehydrogenase (ALDH) family in lysine catabolism. αAAD was low/not detectable in a second GSC subset (n=13) with the same neural metabolic profile as well as in a third GSC subset (n=17) characterized by intense lipid signals. Likewise, αAAD was not detected in the spectra of OB-NPCs or HNPCs. Inhibition of mitochondrial ATP synthase by oligomycin treatment revealed that the lysine degradative pathway leading to αAAD formation proceeds through saccharopine, as usually observed in developing brain. Survival curves indicated that high αAAD levels in GSCs significantly correlated with poor patient survival, similarly to prostate and non-small-cell-lung cancers, where activity of ALDH7A1 correlates with tumor aggressiveness.


Asunto(s)
Ácido 2-Aminoadípico/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patología , Células Madre Neoplásicas/metabolismo , Espectroscopía de Protones por Resonancia Magnética , Adulto , Anciano , Neoplasias Encefálicas/patología , Supervivencia Celular , Femenino , Humanos , Estimación de Kaplan-Meier , Lisina/análogos & derivados , Lisina/metabolismo , Masculino , Redes y Vías Metabólicas , Mitocondrias/metabolismo , Análisis Multivariante , Invasividad Neoplásica , Células Madre Neoplásicas/patología , Procesamiento de Señales Asistido por Computador
7.
BMC Cancer ; 14: 151, 2014 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-24593254

RESUMEN

BACKGROUND: Chemoresistance of glioblastoma multiforme (GBM) has been attributed to the presence within the tumor of cancer stem cells (GSCs). The standard therapy for GBM consists of surgery followed by radiotherapy and the chemotherapeutic agent temozolomide (TMZ). However, TMZ efficacy is limited by O6-methylguanine-DNA-methyltransferase (MGMT) and Mismatch Repair (MMR) functions. Strategies to counteract TMZ resistance include its combination with poly(ADP-ribose) polymerase inhibitors (PARPi), which hamper the repair of N-methylpurines. PARPi are also investigated as monotherapy for tumors with deficiency of homologous recombination (HR). We have investigated whether PARPi may restore GSC sensitivity to TMZ or may be effective as monotherapy. METHODS: Ten human GSC lines were assayed for MMR proteins, MGMT and PARP-1 expression/activity, MGMT promoter methylation and sensitivity to TMZ or PARPi, alone and in combination. Since PTEN defects are frequently detected in GBM and may cause HR dysfunction, PTEN expression was also analyzed. The statistical analysis of the differences in drug sensitivity among the cell lines was performed using the ANOVA and Bonferroni's post-test or the non-parametric Kruskal-Wallis analysis and Dunn's post-test for multiple comparisons. Synergism between TMZ and PARPi was analyzed by the median-effect method of Chou and Talalay. Correlation analyses were done using the Spearman's rank test. RESULTS: All GSCs were MMR-proficient and resistance to TMZ was mainly associated with high MGMT activity or low proliferation rate. MGMT promoter hypermethylation of GSCs correlated both with low MGMT activity/expression (Spearman's test, P = 0.004 and P = 0.01) and with longer overall survival of GBM patients (P = 0.02). Sensitivity of each GSC line to PARPi as single agent did not correlate with PARP-1 or PTEN expression. Notably, PARPi and TMZ combination exerted synergistic antitumor effects in eight out of ten GSC lines and the TMZ dose reduction achieved significantly correlated with the sensitivity of each cell line to PARPi as single agent (P = 0.01). CONCLUSIONS: The combination of TMZ with PARPi may represent a valuable strategy to reverse GSC chemoresistance.


Asunto(s)
Antineoplásicos Alquilantes/farmacología , Dacarbazina/análogos & derivados , Resistencia a Antineoplásicos , Glioblastoma/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Línea Celular Tumoral , Islas de CpG , Metilación de ADN , Dacarbazina/farmacología , Glioblastoma/genética , Glioblastoma/mortalidad , Humanos , O(6)-Metilguanina-ADN Metiltransferasa/genética , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , Regiones Promotoras Genéticas , Temozolomida
8.
Front Immunol ; 8: 147, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28261209

RESUMEN

Stem cell factor (SCF), the ligand of c-kit, is a key cytokine for hematopoiesis. Hematopoietic precursors express c-kit, whereas differentiated cells of hematopoietic lineage are negative for this receptor, with the exception of NK cells, mast cells, and a few others. While it has long been recognized that dendritic cells (DCs) can express c-kit, several questions remain concerning the SCF/c-kit axis in DCs. This is particularly relevant for DCs found in those organs wherein SCF is highly expressed, including the bone marrow (BM). We characterized c-kit expression by conventional DCs (cDCs) from BM and demonstrated a higher proportion of c-kit+ cells among type 1 cDC subsets (cDC1s) than type 2 cDC subsets (cDC2s) in both humans and mice, whereas similar levels of c-kit expression were observed in cDC1s and cDC2s from mouse spleen. To further study c-kit regulation, DCs were generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) from mouse BM, a widely used protocol. CD11c+ cells were purified from pooled non-adherent and slightly adherent cells collected after 7 days of culture, thus obtaining highly purified BM-derived DCs (BMdDCs). BMdDCs contained a small fraction of c-kit+ cells, and by replating them for 2 days with GM-CSF, we obtained a homogeneous population of c-kit+ CD40hi MHCIIhi cells. Not only did BMdDCs express c-kit but they also produced SCF, and both were striking upregulated if GM-CSF was omitted after replating. Furthermore, a small but significant reduction in BMdDC survival was observed upon SCF silencing. Incubation of BMdDCs with SCF did not modulate antigen presentation ability of these cells, nor it did regulate their membrane expression of the chemokine receptor CXCR4. We conclude that the SCF/c-kit-mediated prosurvival circuit may have been overlooked because of the prominent use of GM-CSF in DC cultures in vitro, including those human DC cultures destined for the clinics. We speculate that DCs more prominently rely on SCF in vivo in some microenvironments, with potential implications for graft-versus-host disease and antitumor immunity.

9.
Neuro Oncol ; 19(8): 1097-1108, 2017 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-28204560

RESUMEN

BACKGROUND: Advances from glioma stemlike cell (GSC) research, though increasing our knowledge of glioblastoma (GBM) biology, do not influence clinical decisions yet. We explored the translational power of GSC-enriched cultures from patient-derived tumorspheres (TS) in predicting treatment response. METHODS: The relationship between TS growth and clinical outcome was investigated in 52 GBMs treated with surgical resection followed by radiotherapy and temozolomide (TMZ). The effect on TS of radiation (6 to 60 Gy) and of TMZ (3.9 µM to 1 mM) was related with patients' survival. RESULTS: Generation of TS was an independent factor for poor overall survival (OS) and poor progression-free survival (PFS) (P < .0001 and P = .0010, respectively). Growth rate and clonogenicity of TS predicted poor OS. In general, TS were highly resistant to both radiation and TMZ. Resistance to TMZ was stronger in TS with high clonogenicity and fast growth (P < .02). Shorter PFS was associated with radiation LD50 (lethal dose required to kill 50% of TS cells) >12 Gy of matched TS (P = .0484). A direct relationship was found between sensitivity of TS to TMZ and patients' survival (P = .0167 and P = .0436 for OS and PFS, respectively). Importantly, values for TMZ half-maximal inhibitory concentration <50 µM, which are in the range of plasma levels achieved in vivo, identified cases with longer OS and PFS (P = .0020 and P = .0016, respectively). CONCLUSIONS: Analysis of TS holds translational relevance by predicting the response of parent tumors to radiation and, particularly, to TMZ. Dissecting the clonogenic population from proliferating progeny in TS can guide therapeutic strategies to a more effective drug selection and treatment duration.


Asunto(s)
Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Anciano , Antineoplásicos Alquilantes/uso terapéutico , Neoplasias Encefálicas/patología , Quimioradioterapia/métodos , Dacarbazina/análogos & derivados , Dacarbazina/uso terapéutico , Supervivencia sin Enfermedad , Femenino , Glioblastoma/mortalidad , Glioblastoma/patología , Humanos , Masculino , Persona de Mediana Edad , Temozolomida
10.
Cell Rep ; 17(1): 249-260, 2016 09 27.
Artículo en Inglés | MEDLINE | ID: mdl-27681435

RESUMEN

Estrogen receptor alpha-positive (ERα+) luminal tumors are the most frequent subtype of breast cancer. Stat1(-/-) mice develop mammary tumors that closely recapitulate the biological characteristics of this cancer subtype. To identify transforming events that contribute to tumorigenesis, we performed whole genome sequencing of Stat1(-/-) primary mammary tumors and matched normal tissues. This investigation identified somatic truncating mutations affecting the prolactin receptor (PRLR) in all tumor and no normal samples. Targeted sequencing confirmed the presence of these mutations in precancerous lesions, indicating that this is an early event in tumorigenesis. Functional evaluation of these heterozygous mutations in Stat1(-/-) mouse embryonic fibroblasts showed that co-expression of truncated and wild-type PRLR led to aberrant STAT3 and STAT5 activation downstream of the receptor, cellular transformation in vitro, and tumor formation in vivo. In conclusion, truncating mutations of PRLR promote tumor growth in a model of human ERα+ breast cancer and warrant further investigation.


Asunto(s)
Carcinoma/genética , Receptor alfa de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Animales/genética , Mutación , Receptores de Prolactina/genética , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma/metabolismo , Carcinoma/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Embrión de Mamíferos , Receptor alfa de Estrógeno/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Ratones , Ratones Noqueados , Receptores de Prolactina/metabolismo , Factor de Transcripción STAT1/deficiencia , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Transducción de Señal
11.
Neoplasia ; 15(7): 773-82, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23814489

RESUMEN

Skull base chordomas are challenging tumors due to their deep surgical location and resistance to conventional radiotherapy. Chemotherapy plays a marginal role in the treatment of chordoma resulting from lack of preclinical models due to the difficulty in establishing tumor cell lines and valuable in vivo models. Here, we established a cell line from a recurrent clival chordoma. Cells were cultured for more than 30 passages and the expression of the chordoma cell marker brachyury was monitored using both immunohistochemistry and Western blot. Sensitivity of chordoma cells to the inhibition of specific signaling pathways was assessed through testing of a commercially available small molecule kinase inhibitor library. In vivo tumorigenicity was evaluated by grafting chordoma cells onto immunocompromised mice and established tumor xenografts were treated with rapamycin. Rapamycin was administered to the donor patient and its efficacy was assessed on follow-up neuroimaging. Chordoma cells maintained brachyury expression at late passages and generated xenografts closely mimicking the histology and phenotype of the parental tumor. Rapamycin was identified as an inhibitor of chordoma cell proliferation. Molecular analyses on tumor cells showed activation of the mammalian target of rapamycin signaling pathway and mutation of KRAS gene. Rapamycin was also effective in reducing the growth of chordoma xenografts. On the basis of these results, our patient received rapamycin therapy with about six-fold reduction of the tumor growth rate upon 10-month follow-up neuroimaging. This is the first case of chordoma in whom chemotherapy was tailored on the basis of the sensitivity of patient-derived tumor cells.


Asunto(s)
Antineoplásicos/farmacología , Sirolimus/farmacología , Adulto , Antineoplásicos/uso terapéutico , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cordoma/diagnóstico , Cordoma/tratamiento farmacológico , Cordoma/genética , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Imagen por Resonancia Magnética , Mutación , Cultivo Primario de Células , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras) , Sirolimus/uso terapéutico , Neoplasias de la Base del Cráneo/diagnóstico , Neoplasias de la Base del Cráneo/tratamiento farmacológico , Neoplasias de la Base del Cráneo/genética , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/genética
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