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1.
Int J Mol Sci ; 23(21)2022 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-36361619

RESUMO

Acute myeloid leukemia (AML) and B-cell acute lymphocytic leukemia (B-ALL) are severe blood malignancies affecting both adults and children. Chimeric antigen receptor (CAR)-based immunotherapies have proven highly efficacious in the treatment of leukemia. However, the challenge of the immune escape of cancer cells remains. The development of more affordable and ready-to-use therapies is essential in view of the costly and time-consuming preparation of primary cell-based treatments. In order to promote the antitumor function against AML and B-ALL, we transduced NK-92 cells with CD276-CAR or CD19-CAR constructs. We also attempted to enhance cytotoxicity by a gene knockout of three different inhibitory checkpoints in NK cell function (CBLB, NKG2A, TIGIT) with CRISPR-Cas9 technology. The antileukemic activity of the generated cell lines was tested with calcein and luciferase-based cytotoxicity assays in various leukemia cell lines. Both CAR-NK-92 exhibited targeted cytotoxicity and a significant boost in antileukemic function in comparison to parental NK-92. CRISPR-Cas9 knock-outs did not improve B-ALL cytotoxicity. However, triple knock-out CD276-CAR-NK-92 cells, as well as CBLB or TIGIT knock-out NK-92 cells, showed significantly enhanced cytotoxicity against U-937 or U-937 CD19/tag AML cell lines. These results indicate that the CD19-CAR and CD276-CAR-NK-92 cell lines' cytotoxic performance is suitable for leukemia killing, making them promising off-the-shelf therapeutic candidates. The knock-out of CBLB and TIGIT in NK-92 and CD276-CAR-NK-92 should be further investigated for the treatment of AML.


Assuntos
Leucemia Mieloide Aguda , Linfoma de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Antígenos Quiméricos , Humanos , Antígenos CD19 , Antígenos B7/metabolismo , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Imunoterapia Adotiva/métodos , Células Matadoras Naturais , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo
2.
Pediatr Hematol Oncol ; 38(5): 471-485, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33635158

RESUMO

Arsenic trioxide (ATO) has become an established component of treatment protocols for acute promyelocytic leukemia (APL) with excellent efficacy and no relevant sustained toxicity. Part of its action has been attributed to the inhibition of Hedgehog signaling (Hh) which enables a possible therapeutic approach as many pediatric tumor entities have been associated with increased Hh activity. We retrospectively analyzed 31 patients with refractory and relapsed pediatric cancer who were treated with ATO at the University Children's Hospital of Tuebingen. Additionally a literature review on the clinical and preclinical use of ATO in pediatric cancer treatment was performed.ATO alone as well as combinations with other drugs have proven effective in vitro and in mouse models of various pediatric malignancies. However, only few data on the clinical use of ATO in pediatric patients besides APL exist. In our patient sample, ATO was overall well tolerated in the treatment of various pediatric cancers, even in combination with other cytostatic drugs. Due to distinct tumor entities, differently progressed disease stages and varying co-medication, no clear statement can be made regarding the efficacy of ATO treatment. However, patients with proven Hh activation in molecular tumor profiling surpassed all other patients, who received ATO in an experimental treatment setting, in terms of survival. As molecular profiling of tumors increases and enhanced Hh activity can be detected at an early stage, ATO might expand its clinical use to other pediatric malignancies beyond APL depending on further clinical studies.


Assuntos
Antineoplásicos/uso terapêutico , Trióxido de Arsênio/uso terapêutico , Neoplasias Hematológicas/tratamento farmacológico , Leucemia Promielocítica Aguda/tratamento farmacológico , Adolescente , Adulto , Antineoplásicos/efeitos adversos , Trióxido de Arsênio/efeitos adversos , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Adulto Jovem
3.
Int Arch Allergy Immunol ; 179(2): 123-131, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30909275

RESUMO

BACKGROUND: Liposomal eye spray (LS) has been introduced for the treatment of dry eye. Whether LS can also relieve symptoms due to allergic rhinoconjunctivitis (ARC) has not yet been sufficiently investigated. OBJECTIVES: The aim of this study was to assess the onset of action, the reduction of allergy symptoms, and the safety of LS - a nonpharmacological treatment option - compared with those of antihistamine eye drops (AD). METHODS: In this open, prospective, controlled, monocenter noninterventional study, adults with ARC received either LS or AD for the relief of eye irritation after a positive conjunctival provocation test (CPT). All patients completed a questionnaire before and after the CPT. Eye irritation was rated on a visual analogue scale (VAS) at 6 time points. Conjunctival redness was analyzed using objective digital analysis based on images taken during the study visit. RESULTS: Data were collected from 40 patients (20 per group). In both groups, 80% of patients perceived an onset of action within 0-2 min after application of LS or AD following the CPT. Relief of eye irritation (as determined by VAS) increased throughout the visit for both groups. In the digital analysis, the mean proportion of redness of the eye decreased from 10.3 to 7.0% for LS and from 10.4 to 6.5% for AD, with the largest difference observed 10 min after application (LS: 8.9%; AD: 6.0%; p = 0.094). CONCLUSIONS: LS is a nonpharmacological treatment option for ARC, showing no significant difference or relevant numerical inferiority to AD in any parameter studied. It was generally safe and well tolerated.


Assuntos
Conjuntivite Alérgica/diagnóstico , Conjuntivite Alérgica/tratamento farmacológico , Antagonistas dos Receptores Histamínicos/administração & dosagem , Lipossomos , Soluções Oftálmicas/administração & dosagem , Adulto , Alérgenos/efeitos adversos , Alérgenos/imunologia , Conjuntivite Alérgica/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Índice de Gravidade de Doença , Avaliação de Sintomas , Resultado do Tratamento , Adulto Jovem
4.
Int J Mol Sci ; 19(1)2018 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-29361725

RESUMO

Unlike other malignant bone tumors including osteosarcomas and Ewing sarcomas with a peak incidence in adolescents and young adults, conventional and dedifferentiated chondrosarcomas mainly affect people in the 4th to 7th decade of life. To date, the cell type of chondrosarcoma origin is not clearly defined. However, it seems that mesenchymal stem and progenitor cells (MSPC) in the bone marrow facing a pro-proliferative as well as predominantly chondrogenic differentiation milieu, as is implicated in early stage osteoarthritis (OA) at that age, are the source of chondrosarcoma genesis. But how can MSPC become malignant? Indeed, only one person in 1,000,000 will develop a chondrosarcoma, whereas the incidence of OA is a thousandfold higher. This means a rare coincidence of factors allowing escape from senescence and apoptosis together with induction of angiogenesis and migration is needed to generate a chondrosarcoma. At early stages, chondrosarcomas are still assumed to be an intermediate type of tumor which rarely metastasizes. Unfortunately, advanced stages show a pronounced resistance both against chemo- and radiation-therapy and frequently metastasize. In this review, we elucidate signaling pathways involved in the genesis and therapeutic resistance of chondrosarcomas with a focus on MSPC compared to signaling in articular cartilage (AC).


Assuntos
Neoplasias Ósseas/etiologia , Neoplasias Ósseas/metabolismo , Condrossarcoma/etiologia , Condrossarcoma/metabolismo , Cartilagem Hialina/metabolismo , Cartilagem Hialina/patologia , Animais , Biomarcadores , Neoplasias Ósseas/diagnóstico , Neoplasias Ósseas/terapia , Proliferação de Células , Transformação Celular Neoplásica , Microambiente Celular , Condrogênese , Condrossarcoma/diagnóstico , Condrossarcoma/terapia , Resistencia a Medicamentos Antineoplásicos , Humanos , Hipóxia/metabolismo , Incidência , Células-Tronco Mesenquimais/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Neovascularização Patológica , Células-Tronco/metabolismo
5.
Cell Physiol Biochem ; 43(3): 1301-1308, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28992614

RESUMO

BACKGROUND/AIMS: Rhabdomyosarcoma, the most common pediatric soft tissue sarcoma, may show an intrinsic refractoriness to standard chemotherapy in advanced tumor stages, which is associated with poor prognosis. Cellular mechanisms conferring tumor cell survival and therapy resistance in many tumor types include the serum & glucocorticoid inducible kinase (SGK) 1 pathway, a kinase expressed ubiquitously with particularly strong expression in skeletal muscle and some tumor types. The present study explored whether SGK1 is expressed in rhabdomyosarcoma and, if so, whether this kinase impacts on tumor cell survival, proliferation and migration. Multiple in vitro techniques were used to study the role of SGK1 in rhabdomyosarcoma. METHODS: The Gene Chip® Human Genome U133 Plus 2.0 Array were employed to examine SGK1 transcriptional activity in healthy muscle and rhabdomyosarcoma tissue. SGK1 transcript levels were quantified in rhabdomyosarcoma cell lines RD (embryonal subtype) and RH30 (alveolar subtype) by RT-PCR, cell viability was measured using MTT assays. Clonal cell growth was assessed via colony forming assays and migration experiments were performed in a transwell system. RESULTS: SGK1 is expressed in embryonal and alveolar rhabdomyosarcoma tissue samples and in RD and RH30 rhabdomyosarcoma cell lines. Administration of EMD638683 - an inhibitor specific for SGK1 - decreased viability of RD and RH30 cells, enhanced the effects of the cytotoxic drug doxorubicin leading to reduced migration and decreased cell proliferation. CONCLUSIONS: SGK1 is expressed in rhabdomyosarcoma cells where it contributes to survival, therapy resistance, cell proliferation and migration. Thus, SGK1 inhibitors may be considered a therapeutic option for the treatment of therapy-resistant rhabdomyosarcoma.


Assuntos
Proteínas Imediatamente Precoces/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Antibióticos Antineoplásicos/toxicidade , Benzamidas/toxicidade , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/toxicidade , Humanos , Hidrazinas/toxicidade , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/genética , Músculo Esquelético/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Reação em Cadeia da Polimerase em Tempo Real , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Neoplasias de Tecidos Moles/metabolismo , Neoplasias de Tecidos Moles/patologia
6.
J Neurophysiol ; 115(5): 2330-40, 2016 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-26823514

RESUMO

Ca(2+)-activated potassium currents [IK(Ca)] are an important link between the intracellular signaling system and the membrane potential, which shapes intrinsic electrophysiological properties. To better understand the ionic mechanisms that mediate intrinsic firing properties of olfactory uniglomerular projection neurons (uPNs), we used whole cell patch-clamp recordings in an intact adult brain preparation of the male cockroach Periplaneta americana to analyze IK(Ca) In the insect brain, uPNs form the principal pathway from the antennal lobe to the protocerebrum, where centers for multimodal sensory processing and learning are located. In uPNs the activation of IK(Ca) was clearly voltage and Ca(2+) dependent. Thus under physiological conditions IK(Ca) is strongly dependent on Ca(2+) influx kinetics and on the membrane potential. The biophysical characterization suggests that IK(Ca) is generated by big-conductance (BK) channels. A small-conductance (SK) channel-generated current could not be detected. IK(Ca) was sensitive to charybdotoxin (CTX) and iberiotoxin (IbTX) but not to apamin. The functional role of IK(Ca) was analyzed in occlusion experiments under current clamp, in which portions of IK(Ca) were blocked by CTX or IbTX. Blockade of IK(Ca) showed that IK(Ca) contributes significantly to intrinsic electrophysiological properties such as the action potential waveform and membrane excitability.


Assuntos
Condutos Olfatórios/fisiologia , Canais de Potássio Cálcio-Ativados/metabolismo , Células Receptoras Sensoriais/fisiologia , Potenciais de Ação , Animais , Apamina/farmacologia , Cálcio/metabolismo , Charibdotoxina/farmacologia , Baratas , Masculino , Condutos Olfatórios/citologia , Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Células Receptoras Sensoriais/metabolismo
7.
Int J Cancer ; 135(4): 989-95, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-24420698

RESUMO

Rhabdoid tumors are highly aggressive tumors occurring in infants and very young children. Despite multimodal and intensive therapy prognosis remains poor. Molecular analyses have uncovered several deregulated pathways, among them the CDK4/6-Rb-, the WNT- and the Sonic hedgehog (SHH) pathways. The SHH pathway is activated in rhabdoid tumors by GLI1 overexpression. Here, we demonstrate that arsenic trioxide (ATO) inhibits tumor cell growth of malignant rhabdoid tumors in vitro and in a mouse xenograft model by suppressing Gli1. Our data uncover ATO as a promising therapeutic approach to improve prognosis for rhabdoid tumor patients.


Assuntos
Antineoplásicos/farmacologia , Arsenicais/farmacologia , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/metabolismo , Óxidos/farmacologia , Tumor Rabdoide/tratamento farmacológico , Fatores de Transcrição/metabolismo , Animais , Apoptose , Trióxido de Arsênio , Ciclo Celular , Proliferação de Células , Biologia Computacional , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Camundongos SCID , Transplante de Neoplasias , Prognóstico , Transdução de Sinais , Proteína GLI1 em Dedos de Zinco
8.
J Virol ; 87(6): 3484-501, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23302892

RESUMO

The oncolytic potential of measles vaccine virus (MeV) has been demonstrated in several tumor entities. Here, we investigated the susceptibility of eight sarcoma cell lines to MeV-mediated oncolysis and found five to be susceptible, whereas three proved to be resistant. In the MeV-resistant cell lines, we often observed an inhibition of viral replication along with a strong upregulation of the intracellular virus-sensing molecule RIG-I and of the interferon (IFN)-stimulated gene IFIT1. Not only expression of IFIT1 but also phosphorylation of IFN-stimulated Stat1 took place rapidly and were found to be persistent over time. In contrast, susceptible cell lines showed a much weaker, delayed, or completely missing expression of IFIT1 as well as a delayed or only transient phosphorylation of Stat1, whereas exogenic stimulation with beta interferon (IFN-ß) resulted in a comparable profound activation of Stat1 and expression of IFIT1 in all cell lines. Pretreatment with IFN-ß rendered three of the susceptible cell lines more resistant to MeV-mediated oncolysis. These data suggest that differences in the innate immune defense often account for different degrees of susceptibility of sarcoma cell lines to MeV-mediated oncolysis. From a therapeutic perspective, we were able to overcome resistance to MeV by increasing the multiplicity of infection (MOI) and by addition of the prodrug 5-fluorocytosine (FC), thereby exploiting the suicide gene function of virotherapeutic vector MeV-SCD armed with the SCD fusion protein, which consists of yeast cytosine deaminase and yeast uracil phosphoribosyltransferase.


Assuntos
Vírus do Sarampo/crescimento & desenvolvimento , Vírus do Sarampo/imunologia , Vírus Oncolíticos/crescimento & desenvolvimento , Vírus Oncolíticos/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Humanos , Interferon beta/imunologia , Vírus do Sarampo/fisiologia , Vírus Oncolíticos/fisiologia , Proteínas de Ligação a RNA , Fator de Transcrição STAT1/metabolismo , Replicação Viral
9.
Leuk Res ; 117: 106842, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35490594

RESUMO

Cancer testis antigen PRAME is over-expressed in a variety of malignant cells but is not or minimally expressed in normal non-germ line cells. Adoptive transfer of PRAME-specific T cells is thus under investigation in clinical trials as an innovative therapeutic option for acute myeloid leukemia (AML). However, their senescence-inducing activity has not been studied. This study therefore examines senescence induction in AML cells by PRAME-specific TH1 cells. Analysis of cell cycle and marker expression demonstrate that the supernatants of antigen-stimulated PRAME-specific TH1 cells induce senescence in AML cell lines Kasumi and Nomo-1 through combinative IFN-γ and TNF-α. Additionally IFN-γ and TNF-α secreted by TCR-activated Vδ2+ or CMV-specific T cells can also drive these AML cell lines into terminal growth arrest. G1/0 arrest is also suggested in patient-derived AML by TH1 cytokines or supernatants from Zoledronate-stimulated or aCD3/aCD28-stimulated PBMCs. Thus, we show for the first time that senescence is induced in AML cells by combined IFN-γ and TNF-α, and that these cytokines can be derived either from TCR-engineered CD4+ T cells, or intriguingly from Virus-specific as well as innate Vδ2+ T cells responding to their cognate antigens, namely T-cell responses targeting an antigen that is NOT expressed by the leukemic cells.


Assuntos
Citocinas , Leucemia Mieloide Aguda , Humanos , Interferon gama/metabolismo , Leucemia Mieloide Aguda/terapia , Masculino , Receptores de Antígenos de Linfócitos T , Fator de Necrose Tumoral alfa
10.
Cancers (Basel) ; 13(5)2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33807875

RESUMO

BACKGROUND: Since metastatic spreading of solid tumor cells often leads to a fatal outcome for most cancer patients, new approaches for patient-individualized, targeted immunotherapy are urgently needed. METHODS: Here, we established cell lines from four bone metastases of different tumor entities. We assessed AdCAR NK-92-mediated cytotoxicity in vitro in standard cytotoxicity assays as well as 3D spheroid models Results: AdCAR-engineered NK-92 cells successfully demonstrated distinct and specific cytotoxic potential targeting different tumor antigens expressed on cell lines established from bone metastases of mammary, renal cell and colorectal carcinoma as well as melanomas. In that process AdCAR NK-92 cells produced a multitude of NK effector molecules as well as pro inflammatory cytokines. Furthermore, AdCAR NK-92 showed increased cytotoxicity in 3D spheroid models which can recapitulate in vivo architecture, thereby bridging the gap between in vitro and in vivo models. CONCLUSIONS: AdCAR NK-92 cells may provide an interesting and promising "off-the-shelf" cellular product for the targeted therapy of cancers metastasizing to the bone, while utilization of clinically approved, therapeutic antibodies, as exchangeable adapter molecules can facilitate quick clinical translation.

11.
Cancers (Basel) ; 14(1)2021 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-35008205

RESUMO

Enhancer of Zeste homolog 2 (EZH2) is involved in epigenetic regulation of gene transcription by catalyzing trimethylation of histone 3 at lysine 27. In rhabdomyosarcoma (RMS), increased EZH2 protein levels are associated with poor prognosis and increased metastatic potential, suggesting EZH2 as a therapeutic target. The inhibition of EZH2 can be achieved by direct inhibition which targets only the enzyme activity or by indirect inhibition which also affects activities of other methyltransferases and reduces EZH2 protein abundance. We assessed the direct inhibition of EZH2 by EPZ005687 and the indirect inhibition by 3-deazaneplanocin (DZNep) and adenosine dialdehyde (AdOx) in the embryonal RD and the alveolar RH30 RMS cell line. EPZ005687 was more effective in reducing the cell viability and colony formation, in promoting apoptosis induction, and in arresting cells in the G1 phase of the cell cycle than the indirect inhibitors. DZNep was more effective in decreasing spheroid viability and size in both cell lines than EPZ005687 and AdOx. Both types of inhibitors reduced cell migration of RH30 cells but not of RD cells. The results show that direct and indirect inhibition of EZH2 affect cellular functions differently. The alveolar cell line RH30 is more sensitive to epigenetic intervention than the embryonal cell line RD.

12.
Biomolecules ; 11(11)2021 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-34827559

RESUMO

Mesenchymal glioblastoma stem cells (GSCs), a subpopulation in glioblastoma that are responsible for therapy resistance and tumor spreading in the brain, reportedly upregulate aldehyde dehydrogenase isoform-1A3 (ALDH1A3) which can be inhibited by disulfiram (DSF), an FDA-approved drug formerly prescribed in alcohol use disorder. Reportedly, DSF in combination with Cu2+ ions exerts multiple tumoricidal, chemo- and radio-therapy-sensitizing effects in several tumor entities. The present study aimed to quantify these DSF effects in glioblastoma stem cells in vitro, regarding dependence on ALDH1A3 expression. To this end, two patient-derived GSC cultures with differing ALDH1A3 expression were pretreated (in the presence of CuSO4, 100 nM) with DSF (0 or 100 nM) and the DNA-alkylating agent temozolomide (0 or 30 µM) and then cells were irradiated with a single dose of 0-8 Gy. As read-outs, cell cycle distribution and clonogenic survival were determined by flow cytometry and limited dilution assay, respectively. As a result, DSF modulated cell cycle distribution in both GSC cultures and dramatically decreased clonogenic survival independently of ALDH1A3 expression. This effect was additive to the impairment of clonogenic survival by radiation, but not associated with radiosensitization. Of note, cotreatment with temozolomide blunted the DSF inhibition of clonogenic survival. In conclusion, DSF targets GSCs independent of ALDH1A3 expression, suggesting a therapeutic efficacy also in glioblastomas with low mesenchymal GSC populations. As temozolomide somehow antagonized the DSF effects, strategies for future combination of DSF with the adjuvant standard therapy (fractionated radiotherapy and concomitant temozolomide chemotherapy followed by temozolomide maintenance therapy) are not supported by the present study.


Assuntos
Glioblastoma , Dissulfiram , Reposicionamento de Medicamentos , Temozolomida
13.
Cells ; 10(5)2021 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-33925968

RESUMO

BACKGROUND: Melanoma is the most lethal of all skin-related cancers with incidences continuously rising. Novel therapeutic approaches are urgently needed, especially for the treatment of metastasizing or therapy-resistant melanoma. CAR-modified immune cells have shown excellent results in treating hematological malignancies and might represent a new treatment strategy for refractory melanoma. However, solid tumors pose some obstacles for cellular immunotherapy, including the identification of tumor-specific target antigens, insufficient homing and infiltration of immune cells as well as immune cell dysfunction in the immunosuppressive tumor microenvironment (TME). METHODS: In order to investigate whether CAR NK cell-based immunotherapy can overcome the obstacles posed by the TME in melanoma, we generated CAR NK-92 cells targeting CD276 (B7-H3) which is abundantly expressed in solid tumors, including melanoma, and tested their effectivity in vitro in the presence of low pH, hypoxia and other known factors of the TME influencing anti-tumor responses. Moreover, the CRISPR/Cas9-induced disruption of the inhibitory receptor NKG2A was assessed for its potential enhancement of NK-92-mediated anti-tumor activity. RESULTS: CD276-CAR NK-92 cells induced specific cytolysis of melanoma cell lines while being able to overcome a variety of the immunosuppressive effects normally exerted by the TME. NKG2A knock-out did not further improve CAR NK-92 cell-mediated cytotoxicity. CONCLUSIONS: The strong cytotoxic effect of a CD276-specific CAR in combination with an "off-the-shelf" NK-92 cell line not being impaired by some of the most prominent negative factors of the TME make CD276-CAR NK-92 cells a promising cellular product for the treatment of melanoma and beyond.


Assuntos
Antígenos B7/genética , Regulação Neoplásica da Expressão Gênica , Células Matadoras Naturais/citologia , Melanoma/imunologia , Receptores de Antígenos Quiméricos/genética , Neoplasias Cutâneas/imunologia , Microambiente Tumoral , Antígenos de Neoplasias/imunologia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Movimento Celular , Citotoxicidade Imunológica , Fibroblastos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hipóxia , Sistema Imunitário , Terapia de Imunossupressão , Imunossupressores , Imunoterapia Adotiva/métodos , Técnicas In Vitro , Ácido Láctico/metabolismo , Lentivirus/genética , Melanoma/metabolismo , Invasividade Neoplásica , Neoplasias Cutâneas/metabolismo
14.
Oncoimmunology ; 10(1): 2003532, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35686214

RESUMO

Chimeric antigen receptor (CAR)-T therapy holds great promise to sustainably improve cancer treatment. However, currently, a broad applicability of CAR-T cell therapies is hampered by limited CAR-T cell versatility and tractability and the lack of exclusive target antigens to discriminate cancerous from healthy tissues. To achieve temporal and qualitative control on CAR-T function, we engineered the Adapter CAR (AdCAR) system. AdCAR-T are redirected to surface antigens via biotin-labeled adapter molecules in the context of a specific linker structure, referred to as Linker-Label-Epitope. AdCAR-T execute highly specific and controllable effector function against a multiplicity of target antigens. In mice, AdCAR-T durably eliminate aggressive lymphoma. Importantly, AdCAR-T might prevent antigen evasion by combinatorial simultaneous or sequential targeting of multiple antigens and are capable to identify and differentially lyse cancer cells by integration of adapter molecule-mediated signals based on multiplex antigen expression profiles. In consequence the AdCAR technology enables controllable, flexible, combinatorial, and selective targeting.


Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Animais , Imunoterapia Adotiva , Camundongos , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Linfócitos T , Tecnologia
15.
Cancers (Basel) ; 12(1)2020 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-31947966

RESUMO

Abundant IFN-γ secretion, potent cytotoxicity, and major histocompatibility complex-independent targeting of a large spectrum of tumors make γδ T cells attractive candidates for cancer immunotherapy. Upon tumor recognition through the T-cell receptor (TCR), NK-receptors, or NKG2D, γδ T cells generate the pro-inflammatory cytokines TNF-α and IFN-γ, or granzymes and perforin that mediate cellular apoptosis. Despite these favorable potentials, most clinical trials testing the adoptive transfer of pharmacologically TCR-targeted and expanded γδ T cells resulted in a limited response. Recently, the TCR-independent activation of γδ T cells was identified. However, the modulation of γδ T cell's effector functions solely by cytokines remains to be elucidated. In the present study, we systematically analyzed the impact of IL-2, IL-12, and IL-18 in parallel with TCR stimulation on proliferation, cytokine production, and anti-tumor activity of γδ T cells. Our results demonstrate that IL-12 and IL-18, when combined, constitute the most potent stimulus to enhance anti-tumor activity and induce proliferation and IFN-γ production by γδ T cells in the absence of TCR signaling. Intriguingly, stimulation with IL-12 and IL-18 without TCR stimulus induces a comparable degree of anti-tumor activity in γδ T cells to TCR crosslinking by killing tumor cells and driving cancer cells into senescence. These findings approve the use of IL-12/IL-18-stimulated γδ T cells for adoptive cell therapy to boost anti-tumor activity by γδ T cells.

16.
Int J Oncol ; 57(1): 289-300, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32377699

RESUMO

Rhabdomyosarcoma (RMS) is the most common type of pediatric soft tissue sarcoma. The prognosis of advanced stage RMS remains poor, and metastatic invasion is a major cause of treatment failure. Therefore, there is an urgent need for treatment alternatives focusing on metastatic invasion and drug resistance. The stromal cell­derived factor­1 (SDF­1)/chemokine receptor 4 (CXCR4) axis is a crucial factor for metastatic invasion in RMS. Clinical data has revealed that high CXCR4 expression is associated with a poor outcome and a high metastatic rate in several malignancies, including RMS. Thus, targeting CXCR4 in addition to classical chemotherapy may improve the effectiveness of RMS treatment. In the present study, flow cytometry and reverse transcription­quantitative PCR were used to assess the effects of the combined treatment with a CXCR4 antagonist and chemotherapy on CXCR4 expression in the embryonal RMS (RME) cell line RD and in the alveolar RMS (RMA) cell line RH30. The functional effect of CXCR4 expression on the migratory behavior of RMS cells was analyzed using Transwell assays. Treatment with cytotoxic agents modulated CXCR4 expression in RMS cells in a dose­, drug­ and cell line dependent manner; however, this was not observed in RD cells with vincristine. The expression levels of CXCR4 significantly increased the migratory behavior of RMA and did not affect RME cell migration towards stromal cell­derived factor­1α (SDF­1α). AMD3100 markedly reduced the migration of RH30 cells in the Transwell assays compared with SDF­1α alone, and the cytotoxic agents doxorubicin and vincristine increased this effect. The results of the combined treatment in RMS cells using the CXCR4 antagonist AMD3100 together with cytotoxic drugs demonstrated that this approach may be a promising alternative for the treatment of advanced stage pediatric RMS. The observed effects of circumventing metastatic invasion and drug resistance should be further investigated in vivo.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzilaminas/farmacologia , Ciclamos/farmacologia , Receptores CXCR4/antagonistas & inibidores , Rabdomiossarcoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Benzilaminas/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular , Quimiocina CXCL12/análise , Quimiocina CXCL12/metabolismo , Pré-Escolar , Ciclamos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Lactente , Masculino , Estudos Prospectivos , Receptores CXCR4/análise , Receptores CXCR4/metabolismo , Rabdomiossarcoma/patologia , Transdução de Sinais/efeitos dos fármacos , Vincristina/farmacologia , Vincristina/uso terapêutico
17.
Oncoimmunology ; 9(1): 1825177, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-33457105

RESUMO

Despite the recent success of CAR T cells targeting CD19 and CD22 in hematological malignancies, the production of CAR T cells still requires an extensive manufacturing process. The well-established NK-92 cell line provides a promising alternative to produce CAR-modified effector cells in a GMP-compliant, cost-effective way. NK-92 can be redirected against a variety of surface antigens by our adapter CAR (AdCAR) system utilizing biotinylated antibodies (bAb) as adapter molecules. Selected bAb were capable of inducing significant AdCAR NK-92-mediated lysis of non-Hodgkin lymphoma (NHL) and mantle-cell lymphoma (MCL) cell lines as well as primary MCL and chronic lymphocytic leukemia (CLL) cells. AdCAR specificity was proven using a JeKo-1 CD19/CD20 knockout antigen-loss model. Moreover, through combinations of bAb, AdCAR NK-92 cells are capable of combatting tumor antigen evasion mechanisms. In conclusion, we successfully generated the AdCAR NK-92 cell line which can be manufactured as an "off-the-shelf, on-demand" product allowing universal and tunable tumor targeting.


Assuntos
Receptores de Antígenos Quiméricos , Antígenos CD19 , Linhagem Celular Tumoral , Imunoterapia Adotiva , Células Matadoras Naturais , Receptores de Antígenos Quiméricos/genética
18.
Oncol Rep ; 43(1): 337-345, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31746397

RESUMO

Ewing sarcomas (ES) are highly malignant mesenchymal tumors, which most often occur in children and adolescents. The current treatment of choice comprises wide resection in combination with multimodal chemotherapy including etoposide (Eto). Due to the serious side effects associated with common chemotherapeutics and prevalent multidrug resistance in recurrent and metastatic ES, there is a growing demand for alternative strategies and add­on drugs. Previous research has demonstrated efficient cell death induction by Eto in combination with arsenic trioxide (ATO) in ES cell lines. The aim of the present study was to investigate the effect of different temporal sequences of ATO and Eto administration on apoptosis induction and to explore the effect of both drugs on inhibitory glycogen synthase kinase­3ß (GSK3­ß) phosphorylation as well as multidrug transporter gene expression. The intensity of caspase activation was mainly determined by the Eto doses in A673 and TC­71 cells, whereas in RD­ES cells ATO application actively suppressed Eto­induced apoptosis. This coincided with an increase in inhibitory GSK­3ß phosphorylation in ATO­treated RD­ES cells. Inherent mRNA expression of multidrug resistance­associated protein 1 (MRP1) was low in the ES cell lines compared to that observed in the mesenchymal stem cells (MSC), whereas multidrug resistance protein 1 (MDR1) gene expression was considerably increased in the ES cell lines. ATO treatment reduced MRP1 mRNA expression in the A673 and TC­71 cells, while expression was induced in the MSC and RD­ES cells. In contrast, MDR1 mRNA expression was specifically induced by ATO in the A673 and TC­71 cells, reinforcing the expression differences between MSC and the ES cell lines. Although a reliable cell death induction by the combination of ATO and Eto has been previously shown in ES cell lines, the present study showed marked heterogeneity of the ES cell response to ATO and Eto treatment, illustrating the difficulty of prediction of individual treatment outcome in ES.


Assuntos
Trióxido de Arsênio/farmacologia , Neoplasias Ósseas/metabolismo , Etoposídeo/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Sarcoma de Ewing/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Fosforilação/efeitos dos fármacos , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/genética , Proteína GLI1 em Dedos de Zinco/genética
19.
Oncoimmunology ; 9(1): 1683345, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32002293

RESUMO

Expression of the disialoganglioside GD2 has been identified as a marker antigen associated with a breast cancer stem-like cell (BCSC) phenotype. Here, we report on the evaluation of GD2 as a BCSC-specific target antigen for immunotherapy. GD2 expression was confirmed at variable degree in a set of breast cancer cell lines, predominantly in triple-negative breast cancer (TNBC). To target GD2, we have generated novel anti-GD2 chimeric antigen receptors (GD2-CAR), based on single-chain variable fragments (scFv) derived from the monoclonal antibody (mAb) ch14.18, also known as dinutuximab beta. Expressed on T cells, GD2-CARs mediated specific GD2-dependent T-cell activation and target cell lysis. In contrast to previously described GD2-CARs, no signs of exhaustion by tonic signaling were found. Importantly, application of GD2-CAR expressing T cells (GD2-CAR-T) in an orthotopic xenograft model of TNBC (MDA-MB-231) halted local tumor progression and completely prevented lung metastasis formation. In line with the BCSC model, GD2 expression was only found in a subpopulation (4-6%) of MDA-MB-231 cells before injection. Significant expansion of GD2-CAR-T in tumor-bearing mice as well as T-cell infiltrates in the primary tumor and the lungs were found, indicating site-specific activation of GD2-CAR-T. Our data strongly support previous findings of GD2 as a BCSC-associated antigen. GD2-targeted immunotherapies have been extensively studied in human. In conclusion, GD2-CAR-T should be considered a promising novel approach for GD2-positive breast cancer, especially to eliminate disseminated tumor cells and prevent metastasis formation.


Assuntos
Neoplasias da Mama , Imunoterapia Adotiva , Metástase Neoplásica/prevenção & controle , Células-Tronco Neoplásicas , Receptores de Antígenos Quiméricos , Animais , Neoplasias da Mama/terapia , Feminino , Gangliosídeos , Humanos , Camundongos , Receptores de Antígenos Quiméricos/genética , Linfócitos T
20.
Cells ; 9(12)2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33322555

RESUMO

Sclerosing spindle cell rhabdomyosarcoma (SSRMS) is a rare rhabdomyosarcomas (RMS) subtype. Especially cases bearing a myogenic differentiation 1 (MYOD1) mutation are characterized by a high recurrence and metastasis rate, often leading to a fatal outcome. SSRMS cell lines are valuable in vitro models for studying disease mechanisms and for the preclinical evaluation of new therapeutic approaches. In this study, a cell line established from a primary SSRMS tumor of a 24-year-old female after multimodal chemotherapeutic pretreatment has been characterized in detail, including immunohistochemistry, growth characteristics, cytogenetic analysis, mutation analysis, evaluation of stem cell marker expression, differentiation potential, and tumorigenicity in mice. The cell line which was designated SRH exhibited a complex genomic profile, including several translocations and deletions. Array-comparative genomic hybridization (CGH) revealed an overall predominating loss of gene loci. The mesenchymal tumor origin was underlined by the expression of mesenchymal markers and potential to undergo adipogenic and osteogenic differentiation. Despite myogenic marker expression, terminal myogenic differentiation was inhibited, which might be elicited by the MYOD1 hotspot mutation. In vivo tumorigenicity could be confirmed after subcutaneous injection into NOD/SCID/γcnull mice. Summarized, the SRH cell line is the first adult SSRMS cell line available for preclinical research on this rare RMS subtype.


Assuntos
Genômica , Rabdomiossarcoma/patologia , Adipogenia , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Autenticação de Linhagem Celular/métodos , Hibridização Genômica Comparativa , Feminino , Humanos , Cariotipagem , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína MyoD/genética , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/genética , Transdução de Sinais , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Adulto Jovem
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