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1.
Int J Mol Sci ; 24(6)2023 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-36982873

RESUMO

Tumor-associated carbonic anhydrases IX (CAIX) and XII (CAXII) have long been in the spotlight as potential new targets for anti-cancer therapy. Recently, CAIX/CAXII specific inhibitor SLC-0111 has passed clinical phase I study and showed differential response among patients with colorectal cancer (CRC). CRC can be classified into four different consensus molecular subgroups (CMS) showing unique expression patterns and molecular traits. We questioned whether there is a CMS-related CAIX/CAXII expression pattern in CRC predicting response. As such, we analyzed transcriptomic data of tumor samples for CA9/CA12 expression using Cancertool. Protein expression pattern was examined in preclinical models comprising cell lines, spheroids and xenograft tumors representing the CMS groups. Impact of CAIX/CAXII knockdown and SLC-0111 treatment was investigated in 2D and 3D cell culture. The transcriptomic data revealed a characteristic CMS-related CA9/CA12 expression pattern with pronounced co-expression of both CAs as a typical feature of CMS3 tumors. Protein expression in spheroid- and xenograft tumor tissue clearly differed, ranging from close to none (CMS1) to strong CAIX/CAXII co-expression in CMS3 models (HT29, LS174T). Accordingly, response to SLC-0111 analyzed in the spheroid model ranged from no (CMS1) to clear (CMS3), with moderate in CMS2 and mixed in CMS4. Furthermore, SLC-0111 positively affected impact of single and combined chemotherapeutic treatment of CMS3 spheroids. In addition, combined CAIX/CAXII knockdown and more effective treatment with SLC-0111 reduced clonogenic survival of CMS3 modelling single cells. In conclusion, the preclinical data support the clinical approach of targeted CAIX/CAXII inhibition by showing linkage of expression with response and suggest that patients with CMS3-classified tumors would most benefit from such treatment.


Assuntos
Anidrases Carbônicas , Neoplasias Colorretais , Humanos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Anidrase Carbônica IX/genética , Anidrase Carbônica IX/metabolismo , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Compostos de Fenilureia , Sulfonamidas , Animais
2.
Cytotherapy ; 17(2): 186-98, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25593077

RESUMO

BACKGROUND AIMS: The immunomodulating capacity of multipotent mesenchymal stromal cells (MSCs) qualifies them as a therapeutic tool in several diseases. However, repeated transplantation with products of reproducible characteristics may be required. This could be achieved with cryopreserved aliquots of Good Manufacturing Practice (GMP)-grade MSCs. However, the impact of cryopreservation on the characteristics of GMP-MSCs is ill defined. METHODS: We produced fresh and cryopreserved MSCs from human donors with a xenogen-free GMP protocol. Immunogenicity and immunomodulating capacity were tested in co-culture with putative recipient-specific peripheral blood mononuclear cells (PBMCs). Risk of malignant transformation was assessed in vitro and in vivo. RESULTS: Cryopreservation had no impact on viability and consensus criteria of MSCs. In co-culture with PBMCs, MSCs showed low immunogenicity and suppressed mitogen-stimulated proliferation of PBMC irrespective of cryopreservation. Cytogenetic aberrations were not observed consistently in fresh and cryopreserved products, and no signs of malignant transformation occurred in functional assays. MSC products from an elderly pretreated donor showed reduced functional quality, but imminent failure of functional criteria could be detected by an increased population doubling time in early passages. DISCUSSION: This study is the first systematic analysis on cryopreservation of xenogen-free human bone marrow-derived GMP-MSCs. The data support that cryopreservation does not alter the characteristics of the cells and thus may allow the generation of products for serial transplantation. In addition, the protocol allowed early detection of MSC products with low functional capacity.


Assuntos
Transformação Celular Neoplásica/imunologia , Criopreservação , Imunomodulação/imunologia , Células-Tronco Mesenquimais/imunologia , Células-Tronco Pluripotentes/imunologia , Adulto , Idoso , Diferenciação Celular/imunologia , Proliferação de Células , Sobrevivência Celular , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Técnicas de Cocultura , Feminino , Fidelidade a Diretrizes , Humanos , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Células-Tronco Pluripotentes/citologia , Controle de Qualidade , Adulto Jovem
3.
Cancers (Basel) ; 15(16)2023 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-37627150

RESUMO

Colorectal cancer (CRC) is a heterogeneous disease. More insight into the biological diversity of CRC is needed to improve therapeutic outcomes. Established CRC cell lines are frequently used and were shown to be representative models of the main subtypes of CRC at the genomic and transcriptomic level. In the present work, we established stable, luciferase expressing derivatives from 10 well-established CRC cell lines, generated spheroids and subcutaneous xenograft tumors in nude mice, and performed comparative characterization of these model systems. Transcriptomic analyses revealed the close relation of cell lines with their derived spheroids and xenograft tumors. The preclinical model systems clustered with patient tumor samples when compared to normal tissue thereby confirming that cell-line-based tumor models retain specific characteristics of primary tumors. Xenografts showed different differentiation patterns and bioluminescence imaging revealed metastatic spread to the lungs. In addition, the models were classified according to the CMS classification system, with further sub-classification according to the recently identified two intrinsic epithelial tumor cell states of CRC, iCMS2 and iCMS3. The combined data showed that regarding primary tumor characteristics, 3D-spheroid cultures resemble xenografts more closely than 2D-cultured cells do. Furthermore, we set up a bioluminescence-based spheroid cytotoxicity assay in order to be able to perform dose-response relationship studies in analogy to typical monolayer assays. Applying the established assay, we studied the efficacy of oxaliplatin. Seven of the ten used cell lines showed a significant reduction in the response to oxaliplatin in the 3D-spheroid model compared to the 2D-monolayer model. Therapy studies in selected xenograft models confirmed the response or lack of response to oxaliplatin treatment. Analyses of differentially expressed genes in these models identified CAV1 as a possible marker of oxaliplatin resistance. In conclusion, we established a combined 2D/3D, in vitro/in vivo model system representing the heterogeneity of CRC, which can be used in preclinical research applications.

4.
J Cell Mol Med ; 14(9): 2292-304, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19508388

RESUMO

Colorectal carcinoma (CRC) constitutes a common malignancy with limited therapeutic options in metastasized stages. Mesenchymal stem cells (MSC) home to tumours and may therefore serve as a novel therapeutic tool for intratumoral delivery of antineoplastic factors. Tumour necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) which promises apoptosis induction preferentially in tumour cells represents such a factor. We generated TRAIL-MSC by transduction of human MSC with a third generation lentiviral vector system and analysed their characteristics and capacity to inhibit CRC growth. (1) TRAIL-MSC showed stable transgene expression with neither changes in the defining MSC characteristics nor signs of malignant transformation. (2) Upon direct in vitro coculture TRAIL-MSC induced apoptosis in TRAIL-sensitive CRC-cell lines (DLD-1 and HCT-15) but also in CRC-cell lines resistant to soluble TRAIL (HCT-8 and SW480). (3) In mixed subcutaneous (s.c.) xenografts TRAIL-MSC inhibited CRC-tumour growth presumably by apoptosis induction but a substantial proportion of TRAIL-MSC within the total tumour cell number was needed to yield such anti-tumour effect. (4) Systemic application of TRAIL-MSC had no effect on the growth of s.c. DLD-1 xenografts which appeared to be due to a pulmonary entrapment and low rate of tumour integration of TRAIL-MSC. Systemic TRAIL-MSC caused no toxicity in this model. (5) Wild-type MSC seemed to exert a tumour growth-supporting effect in mixed s.c. DLD-1 xenografts. These novel results support the idea that lentiviral TRAIL-transgenic human MSC may serve as vehicles for clinical tumour therapy but also highlight the need for further investigations to improve tumour integration of transgenic MSC and to clarify a potential tumour-supporting effect by MSC.


Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Lentivirus/genética , Células-Tronco Mesenquimais/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Transgenes/genética , Animais , Apoptose , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/patologia , Resistencia a Medicamentos Antineoplásicos , Humanos , Transplante de Células-Tronco Mesenquimais , Camundongos , Camundongos Nus , Solubilidade , Transdução Genética , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Int J Gynecol Cancer ; 19(8): 1446-53, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20009905

RESUMO

BACKGROUND: This phase 2 trial was designed to evaluate the efficacy and toxicity of a combination of docetaxel, gemcitabine, and oxaliplatin for platinum- and paclitaxel-pretreated epithelial ovarian cancer. PATIENTS AND METHODS: Heavily pretreated patients (N = 30; median age, 61 years) received docetaxel, 55 mg/m2; gemcitabine, 500 mg/m2 (day 1); and oxaliplatin, 70 mg/m2 (day 2) biweekly. Twelve patients had platinum-sensitive disease, and 18 patients had platinum-resistant disease. RESULTS: Median follow-up was 18.6 months. No differences in patient characteristics were observed between patients with carboplatinum-sensitive and carboplatinum-resistant disease. In patients with carboplatin-sensitive disease, an overall response (OR) of 83.3%, a progression-free survival of 10.6 months, and an overall survival of 18.9 months were observed. In patients with carboplatinum-resistant disease, an OR was seen in 38.9% with a progression-free survival of 5.3 months and an overall survival of 16.3 months. Patients with platinum-refractory disease (progression under previous carboplatinum therapy, n = 13) had an OR of 23%, whereas patients with objective response but relapse less than 6 months after carboplatinum therapy had an OR of 80.0%. Grade 3 and 4 toxicities were only observed for anemia (6.7%), neutropenia (20.0%), thrombopenia, peripheral neuropathy, and diarrhea (16.7%). No neutropenic fever or treatment-related death occurred. CONCLUSIONS: In comparison with current standard protocols, a combination of docetaxel, gemcitabine, and oxaliplatin showed considerably higher efficacy without remarkable increased toxicity; particularly for patients with early relapse after a platinum-containing therapy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias das Tubas Uterinas/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Peritoneais/tratamento farmacológico , Adolescente , Adulto , Idoso , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Docetaxel , Neoplasias das Tubas Uterinas/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Compostos Organoplatínicos/administração & dosagem , Neoplasias Ovarianas/patologia , Oxaliplatina , Paclitaxel/administração & dosagem , Neoplasias Peritoneais/patologia , Prognóstico , Estudos Prospectivos , Fatores de Risco , Taxoides/administração & dosagem , Resultado do Tratamento , Adulto Jovem , Gencitabina
6.
Cell Cycle ; 8(6): 916-24, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19221495

RESUMO

Differentiation and malignant transformation of stem cells are regulated by epigenetic mechanisms. We analyzed promoter methylation and expression of the stem cell determining genes Brachyury, DPPA5, FGF4, FOXD3, LIN28, NESTIN and ZFP42 depending on the differentiation state in human mesenchymal stem cells (MSC), human embryonal carcinoma cells (ECC) and somatic tumor cells. Differentiation of MSC into osteoblasts and adipocytes was accompanied with a loss of expression of the Brachyury gene and downregulation of LIN28. Inactivation of Brachyury was associated with progressive methylation of its CpG island promoter. In ECC promoter methylation of stem cell markers was more frequent in the differentiated subgroup (71%) compared to undifferentiated ECC (29%) and this was associated with downregulation of Brachyury, DPPA5, FGF4, FOXD3, LIN28 and ZFP42. DPPA5 was methylated and NESTIN was unmethylated in most tumor cells. In somatic tumor cells, methylation of stem cell markers (Brachyury, DPPA5, FGF4, FOXD3, LIN28 and ZFP42) was frequently observed (85%). Treatment of cell lines with an inhibitor of DNA methyltransferase reactivated the expression of DPPA5, FGF4, FOXD3, LIN28 and ZFP42, indicating that aberrant promoter methylation is a crucial event that results in their silencing. Our results suggest that epigenetic inactivation of stem cell associated genes is mediated by promoter methylation and that this may represent a fundamental mechanism during normal differentiation processes.


Assuntos
Diferenciação Celular/genética , Ilhas de CpG , Metilação de DNA , Inativação Gênica , Células-Tronco Mesenquimais/metabolismo , Regiões Promotoras Genéticas/genética , Linhagem Celular Tumoral , Humanos , Células-Tronco Mesenquimais/citologia
7.
Eur J Haematol ; 78(6): 527-31, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17509106

RESUMO

OBJECTIVE: Toxicity related to the infusion of dimethylsulfoxide (DMSO)-cryopreserved peripheral blood stem cells (DMSO-PBSC) mainly comprises cardiovascular events. Fatal neurotoxicity has been reported in a few cases. DMSO represents the putative causative agent of such rare toxicities and elaborate strategies to replace DMSO would benefit from the identification of predisposing factors for DMSO-related toxicities. METHODS: Here, we report on DMSO-related neurotoxicity in a series of patients (n = 51) receiving DMSO-PBSC. The analyzed patient-series included eight patients with pre-existing cerebral disease, partially with a history of epileptic seizures. RESULTS: Neurotoxicity was observed in only one patient who suffered from a generalized tonic seizure upon infusion of DMSO-PBSC and for which the clinical course is reported herein. No neurotoxicity was observed in the group of patients with pre-existing neurological disease. Furthermore, no neurotoxicity was observed in patients who received particularly large volumes of DMSO. In all patients, mild non-neurological side effects occurred but besides the reported seizure no other severe adverse events were observed upon PBSC-infusion. CONCLUSIONS: To our knowledge, this is the first report addressing the identification of predisposing factors for DMSO-related neurotoxicty. We conclude that infusion of DMSO-PBSC can be performed safely in patients with pre-existing cerebral disease despite the rare occurrence of severe neurotoxicity. Retrospective multicenter studies are warranted to identify patients who would benefit from elaborate methods of DMSO-replacement.


Assuntos
Encefalopatias/terapia , Dimetil Sulfóxido/toxicidade , Transplante de Células-Tronco , Adulto , Idoso , Criopreservação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
8.
Gut ; 56(3): 405-15, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16928726

RESUMO

AIMS: At present, clinical success of hepatocyte transplantation as an alternative to whole liver transplantation is hampered by the limited availability of suitable donor organs for the isolation of transplantable hepatocytes. Hence, novel cell sources are required to deliver hepatocytes of adequate quality for clinical use. Mesenchymal stem cells (MSCs) from human bone marrow may have the potential to differentiate into hepatocytes in vitro and in vivo. METHODS: Isolated MSCs were selected by density gradient centrifugation and plastic adherence, differentiated in the presence of human hepatocyte growth medium and transplanted in immunodeficient Pfp/Rag2 mice. RESULTS: Here, we demonstrate that human MSCs gain in vitro the characteristic morphology and function of hepatocytes in response to specified growth factors. Specifically, preconditioned MSCs store glycogen, synthesise urea and feature the active hepatocyte-specific gene promoter of phosphoenolpyruvate carboxykinase (PCK1). After transplantation into livers of immunodeficient mice, preconditioned MSCs engraft predominantly in the periportal portion of the liver lobule. In situ, the cells continue to store glycogen and express PCK1, connexin32, albumin and the human hepatocyte-specific antigen HepPar1, indicating that the transplanted cells retain prominent qualities of hepatocytes after their regional integration. CONCLUSION: MSCs derived from human bone marrow may serve as a novel source for the propagation of hepatocyte-like cells suitable for cell therapy in liver diseases.


Assuntos
Hepatócitos/citologia , Fígado/citologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Animais , Diferenciação Celular , Células Cultivadas , Meios de Cultivo Condicionados , Sobrevivência de Enxerto , Hepatócitos/fisiologia , Humanos , Técnicas Imunoenzimáticas , Hibridização in Situ Fluorescente , Fígado/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Condicionamento Pré-Transplante/métodos , Transplante Heterólogo
9.
Stem Cells ; 24(12): 2753-65, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16931776

RESUMO

For various potential clinical applications, the use of autologous human MSCs (hMSCs) would be favorable. In vitro observations suggested that hMSCs are resistant for chemotherapeutic substances; however, no data exist on the characteristics of hMSCs from bone marrow (BM) of chemotherapeutically treated patients. Here, we analyzed the character of hMSCs derived from chemotherapy-exposed BM and the in vitro response of hMSCs to chemotherapeutic substances. Colony-forming units-fibroblast (CFU-Fs) were isolated from BM aspirates of patients after high-dose or standard chemotherapy and of donors with unaffected BM. CFU-Fs from chemotherapy-exposed and unaffected BM contained hMSCs with similar phenotype, proliferation capacity, and differentiation potential. No obvious influence of age, sex, or time since chemotherapy exposure on the presence and characteristics of hMSCs was observed. In vitro, hMSCs showed a significant resistance for cisplatin, vincristine, and etoposide compared with sensitive tumor cell lines, particularly at apoptosis-inducing doses. The phenotype and differentiation potential of hMSCs was not altered by genotoxic treatment under clinically relevant conditions in vitro. hMSCs showed an elevated threshold for cisplatin-induced apoptosis, which was characterized by a lack of caspase-9 activity in apoptotic cells. In vitro exposure of hMSCs to cisplatin, vincristine, and etoposide resulted in an increased p53 expression, independent of apoptosis induction. We conclude that hMSCs can be isolated from chemotherapy-exposed BM in sufficient number and quality for potential clinical applications in chemotherapeutically treated patients. Our data suggest that an elevated apoptotic threshold contributes not only to the persistence of hMSCs in the BM after chemotherapy but also to their lifelong presence in the adult BM.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Resistência a Medicamentos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Adulto , Idoso , Caspase 9/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Separação Celular , Ativação Enzimática/efeitos dos fármacos , Feminino , Fibroblastos/citologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutagênicos , Fenótipo , Proteína Supressora de Tumor p53/efeitos dos fármacos
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