RESUMEN
The treatment response to anti-angiogenic agents varies among cancer patients and predictive biomarkers are needed to identify patients with resistant cancer or guide the choice of anti-angiogenic treatment. We present "the Cancer Angiogenesis Co-Culture (CACC) assay", an in vitro Functional Precision Medicine assay which enables the study of tumouroid induced angiogenesis. This assay can quantify the ability of a patient-derived tumouroid to induce vascularization by measuring the induction of tube formation in a co-culture of vascular cells and tumoroids established from the primary colorectal tumour or a metastasis. Furthermore, the assay can quantify the sensitivity of patient-derived tumoroids to anti-angiogenic therapies. We observed that tube formation increased in a dose-dependent manner upon treatment with the pro-angiogenic factor vascular endothelial growth factor A (VEGF-A). When investigating the angiogenic potential of tumoroids from 12 patients we found that 9 tumoroid cultures induced a significant increase in tube formation compared to controls without tumoroids. In these 9 angiogenic tumoroid cultures the tube formation could be abolished by treatment with one or more of the investigated anti-angiogenic agents. The 3 non-angiogenic tumoroid cultures secreted VEGF-A but we observed no correlation between the amount of tube formation and tumoroid-secreted VEGF-A. Our data suggests that the CACC assay recapitulates the complexity of tumour angiogenesis, and when clinically verified, could prove a valuable tool to quantify sensitivity towards different anti-angiogenic agents.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Técnicas de Cocultivo/métodos , Neovascularización Patológica/tratamiento farmacológico , Anciano , Anciano de 80 o más Años , Inductores de la Angiogénesis/farmacología , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Femenino , Factores de Crecimiento de Fibroblastos/farmacología , Fibroblastos/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Factor de Crecimiento Derivado de Plaquetas/farmacología , Esferoides Celulares/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
BACKGROUND: Pre-therapeutic analysis of three-dimensional spheroid cultures of primary tumour samples is a promising approach of assessing susceptibility to potential treatment. The phosphatidylinositol-3-kinase/AKT serine/threonine kinase/mammalian target of rapamycin (PI3K/AKT/mTOR) signalling pathway is frequently activated in colorectal cancer (CRC). In previous work, we showed combined inhibition of AKT and mTOR to be highly synergistic in cell lines from patients with hepatocellular carcinoma and cholangiocarcinoma in vitro as well as in vivo in murine xenograft tumour models. MATERIALS AND METHODS: Patient-derived xenograft colorectal carcinoma cell lines HROC80 T1 M1, HROC147 T0 M1, HROC147Met, HROC277 T0 M1 and HROC277Met2 were treated with AKT inhibitor MK2206, mTOR inhibitor RAD001 or the combination of both drugs. The sensitivity of these cell lines to inhibition was evaluated by calculation of combinatory indices after bromodeoxyuridine assays and analysis of the respective pathways by western blotting. Furthermore, the dual inhibition of AKT and mTOR was confirmed in vivo in a xenograft mouse model. Additionally, primary CRC samples of four patients were embedded in a three-dimensional matrix and the sensitivity of these samples was analyzed by measurement of the spheroid area. RESULTS: In this study, we demonstrate that combined treatment with MK2206 and RAD001 resulted in strong synergistic effects on growth of several primary CRC cell lines and reduced the growth of a patient-derived CRC xenograft in a xenotransplantation mouse model in vivo. Interestingly, the response to treatment varied between cell lines derived from the primary lesion and a liver metastasis of the same patient. In addition, combined treatment with AKT and mTOR inhibitors resulted in a synergistic inhibition of tumouroid growth in all four of the primary patient samples, analyzed in a three-dimensional spheroid model in vitro. CONCLUSION: Our data demonstrate that combined treatment with AKT and mTOR inhibitors exhibits synergistic effects on proliferation of cell lines and primary tumour cells from patients with CRC and may be a promising approach for the treatment of CRC.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Esferoides Celulares/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Everolimus/administración & dosificación , Compuestos Heterocíclicos con 3 Anillos/administración & dosificación , Humanos , Ratones Endogámicos , Ratones Desnudos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Esferoides Celulares/patología , Serina-Treonina Quinasas TOR/metabolismo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
The aim of the present study was to characterize a patient-derived in vitro 3D model (ie tumoroid) established from colorectal adenocarcinoma. This study investigated the growth rate of tumoroids and whether the Kirsten rat sarcoma (KRAS) mutations in the parental tumour accelerate this rate. The tumoroids were established from surgical resections of primary and metastatic colorectal adenocarcinoma from 26 patients. The in vitro growth rate of these tumoroids was monitored by automated imaging and recorded as relative growth rate. The KRAS hotspot mutations were investigated on the parental tumours by Ion Torrent™ next-generation sequencing. The KRAS mutations were detected in 58% of the parental tumours, and a significantly higher growth rate was observed for tumoroids established from the KRAS-mutated tumours compared to wild-type tumours (P < 0.0001). The average relative growth rate (log10) on day 10 was 0.360 ± 0.180 (mean ± SD) for the KRAS-mutated group and 0.098 ± 0.135 (mean ± SD) for the KRAS wild-type group. These results showed that the presence of KRAS mutations in parental tumours is associated with an acceleration of the growth rate of tumoroids. The future perspective for such a model could be the implementation of chemoassays for personalized medicine.
Asunto(s)
Adenocarcinoma/genética , Adenocarcinoma/secundario , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Mutación , Proteínas Proto-Oncogénicas p21(ras)/genética , Anciano , Anciano de 80 o más Años , Análisis Mutacional de ADN/métodos , Femenino , Predisposición Genética a la Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Organoides , Fenotipo , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Chemotherapy treatment of cancer remains a challenge due to the molecular and functional heterogeneity displayed by tumours originating from the same cell type. The pronounced heterogeneity makes it difficult for oncologists to devise an effective therapeutic strategy for the patient. One approach for increasing treatment efficacy is to test the chemosensitivity of cancer cells obtained from the patient's tumour. 3D culture represents a promising method for modelling patient tumours in vitro. The aim of this study was therefore to evaluate how closely short-term spheroid cultures of primary colorectal cancer cells resemble the original tumour. Colorectal cancer cells were isolated from human tumour tissue and cultured as spheroids. Spheroid cultures were established with a high success rate and remained viable for at least 10 days. The spheroids exhibited significant growth over a period of 7 days and no difference in growth rate was observed for spheroids of different sizes. Comparison of spheroids with the original tumour revealed that spheroid culture generally preserved adenocarcinoma histology and expression patterns of cytokeratin 20 and carcinoembryonic antigen. Interestingly, spheroids had a tendency to resemble tumour protein expression more closely after 10 days of culture compared to 3 days. Chemosensitivity screening using spheroids from five patients demonstrated individual response profiles. This indicates that the spheroids maintained patient-to-patient differences in sensitivity towards the drugs and combinations most commonly used for treatment of colorectal cancer. In summary, short-term spheroid culture of primary colorectal adenocarcinoma cells represents a promising in vitro model for use in personalized medicine.
Asunto(s)
Neoplasias Colorrectales/patología , Modelos Biológicos , Medicina de Precisión , Esferoides Celulares/patología , Adenocarcinoma/patología , Proliferación Celular , Supervivencia Celular , Células Epiteliales/patología , Fibroblastos/patología , Humanos , Queratina-20/metabolismo , Proteínas de Neoplasias/metabolismo , Células Tumorales CultivadasRESUMEN
The structure of D-amino acid hexapeptides that promote cellular adhesion was determined by screening D-amino acid hexapeptide libraries synthesized on otherwise inert beaded PEGA resin. These new adhesion molecules provide a completely stable cellular environment and facilitate the maintenance of a monolayer of cells on beads for extended periods. The presence of the peptides promotes spreading of the cells on the bead surface. Not surprisingly, the molecules contained a significant number of arginines and/or lysines. However, the exact structure of each peptide is quite important for the degree of adhesion observed, and a motif with three or four basic amino acids spaced within amino acids of intermediate polarity clearly prevailed, for example, k-l/r-h-r-i/v-r-a; this maintains a polar/hydrophobic balance.
Asunto(s)
Oligopéptidos/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Adhesión Celular , Línea Celular , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Simulación de Dinámica Molecular , Oligopéptidos/química , Biblioteca de Péptidos , Polietilenglicoles/químicaRESUMEN
Apoptotic induction mechanisms are of crucial importance for the general homeostasis of multicellular organisms. In cancer the apoptotic pathways are downregulated, which, at least partly, is due to an abundance of inhibitors of apoptosis proteins (IAPs) that block the apoptotic cascade by deactivating proteolytic caspases. The Smac protein has an antagonistic effect on IAPs, thus providing structural clues for the synthesis of new pro-apoptotic compounds. Herein, we report a solid-phase approach for the synthesis of Smac-derived tetrapeptide libraries. On the basis of a common (N-Me)AVPF sequence, peptides incorporating triazoloprolines and biarylalanines were synthesized by means of Cu(I)-catalyzed azide-alkyne cycloaddition and Pd-catalyzed Suzuki cross-coupling reactions. Solid-phase procedures were optimized to high efficiency, thus accessing all products in excellent crude purities and yields (both typically above 90%). The peptides were subjected to biological evaluation in a live/dead cellular assay which revealed that structural decorations on the AVPF sequence indeed are highly important for cytotoxicity toward HeLa cells.
Asunto(s)
Alanina/química , Proteínas Inhibidoras de la Apoptosis/síntesis química , Oligopéptidos/síntesis química , Peptidomiméticos/síntesis química , Prolina/química , Técnicas de Síntesis en Fase Sólida/métodos , Alanina/análogos & derivados , Alquinos/química , Azidas/química , Catálisis , Cobre/química , Hidrocarburos Aromáticos/química , Proteínas Inhibidoras de la Apoptosis/química , Proteínas Inhibidoras de la Apoptosis/farmacología , Modelos Químicos , Oligopéptidos/química , Oligopéptidos/farmacología , Paladio/química , Peptidomiméticos/química , Peptidomiméticos/farmacología , Prolina/análogos & derivados , Triazoles/químicaRESUMEN
A huge unleashed potential lies hidden in the large and diverse pool of encoded and particularly nonencoded chiral alpha-, beta-, and gamma-amino acids available today. Although these have been extensively exploited in peptide science, the community of organic chemistry has only used this source of diversity in a quite focused and targeted manner. The properties and behavior of peptides as functional molecules in biology are well documented and based on the ability of peptides to adapt a range of discrete conformers at a minimal entropic penalty and therefore ideally fitting their endogenous targets. The development of new organic reactions and chemistries that in a general and quantitative way transform peptides into new functional molecules, preferably on solid support, is a source of completely new classes of molecules with important and advantageous functional properties. The peptide diversity and the ability to perform chemistry on solid support add tremendously to the combinatorial scope of such reactions in pharmaceutical and materials screening scenario. In recent years, the need for "click" reactions to shape complex molecular architecture has been realized mainly with a basis in the world of peptides and DNA, and in polymer chemistry where connection of highly functionalized biologically active substances or property bearing fragments are assembled as molecular LEGO using quantitative and orthogonal click chemistries. In this article, three such new reactions originating in the Carlsberg Laboratory over the last decade taking advantage of organic transformations in the peptide framework is presented. Initially, the click reaction between azide and terminal alkynes catalyzed by Cu(1) (CuAAC-reaction) is described. This CuAAC "click" reaction was observed first at Carlsberg Laboratory in reactions of azido acid chlorides with alkynes on solid support. Second, the Electrophilic Aromatic Substitution Cyclization-Intramolecular Click-Cascade (EASCy-ICC) reaction will be presented. This quantitative stereo-selective cascade reaction provides a highly diverse set of interesting novel scaffolds from peptides. Finally, we describe the preparation of solid phase peptide phosphine- and carbene-based green catalysts (organozymes), which upon complex formation with transition metal perform with high turnovers under aqueous conditions. These catalysts thrive from the peptide folding and diversity, while phosphines and carbenes in the backbone provide for bidental complex formation with transition metals in a format providing an excellent entry into combinatorial catalyst chemistry.