Project description:Altered lipid metabolism is one of the hallmarks of cancer. Cellular proliferation and de novo synthesis of lipids are related to cancer progression. In this study, we evaluated the lipidomic profile of two-dimensional (2D) monolayer and multicellular tumor spheroids from the HCT 116 colon carcinoma cell line. We utilized serial trypsinization on the spheroid samples to generate three cellular populations representing the proliferative, quiescent, and necrotic regions of the spheroid. This analysis enabled a comprehensive identification and quantification of lipids produced in each of the spheroid layer and 2D cultures. We show that lipid subclasses associated with lipid droplets form in oxygen-restricted and acidic regions of spheroids and are produced at higher levels than in 2D cultures. Additionally, sphingolipid production, which is implicated in cell death and survival pathways, is higher in spheroids relative to 2D cells. Finally, we show that increased numbers of lipids composed of polyunsaturated fatty acids (PUFAs) are produced in the quiescent and necrotic regions of the spheroid. The lipidomic signature for each region and cell culture type highlights the importance of understanding the spatial aspects of cancer biology. These results provide additional lipid biomarkers in colon cancer cells that can be further studied to target pivotal lipid production pathways.
Project description:Cell cultures are widely used model systems. Some immortalized cell lines can be grown in either two-dimensional (2D) adherent monolayers or in three-dimensional (3D) multicellular aggregates, or spheroids. Here, the quantitative proteome and phosphoproteome of colon carcinoma HT29 cells cultures in 2D monolayers and 3D spheroids were compared with a stable isotope labeling of amino acids (SILAC) labeling strategy. Two biological replicates from each sample were examined, and notable differences in both the proteome and the phosphoproteome were determined by nanoliquid chromatography tandem mass spectrometry (LC-MS/MS) to assess how growth configuration affects molecular expression. A total of 5867 protein groups, including 2523 phosphoprotein groups and 8733 phosphopeptides were identified in the samples. The Gene Ontology analysis revealed enriched GO terms in the 3D samples for RNA binding, nucleic acid binding, enzyme binding, cytoskeletal protein binding, and histone binding for their molecular functions (MF) and in the process of cell cycle, cytoskeleton organization, and DNA metabolic process for the biological process (BP). The KEGG pathway analysis indicated that 3D cultures are enriched for oxidative phosphorylation pathways, metabolic pathways, peroxisome pathways, and biosynthesis of amino acids. In contrast, analysis of the phosphoproteomes indicated that 3D cultures have decreased phosphorylation correlating with slower growth rates and lower cell-to-extracellular matrix interactions. In sum, these results provide quantitative assessments of the effects on the proteome and phosphoproteome of culturing cells in 2D versus 3D cell culture configurations.
Project description:The progression of cancer cell migration, invasion and subsequent metastasis is the main cause of mortality in cancer patients. Through creating more accurate cancer models, we can achieve more precise results, which will lead to a better understanding of the invasion process. This holds promise for more effective prevention and treatment strategies. Although numerous 2D and 3D cell culture systems have been developed, they poorly reflect the in vivo situation and many questions have remained unanswered. This work describes a novel dynamic 3D cell culture system aimed at advancing our comprehension of cancer cell migration. With the newly designed cultivation chamber, 3D tumor spheroids were cultivated within a collagen I matrix in the presence of fluid flow to study the migration of cancer cells from spheroids in the matrix. Using light sheet microscopy and histology, we demonstrated that the morphology of spheroids is influenced by dynamic culture and that, in contrast to static culture, spheroids in dynamic culture are characterized by the absence of a large necrotic core. Additionally, this influence extends to an increase in the size of migration area, coupled with an increase in expression of some genes related to epithelial-mesenchymal transition (EMT). The results here highlight the importance of dynamic culture in cancer research. Although the dynamic 3D cell culture system in this study was used to investigate migration of one cell type into a matrix, it has the potential to be further developed and used for more complex models consisting of different cell types or to analyze other steps of metastasis development such as transendothelial migration or extravasation.
Project description:Colorectal cancer demonstrates intra-tumour heterogeneity formed by a hierarchical structure comprised of cancer stem cells (CSCs) and their differentiated progenies. The mechanism by which CSCs are maintained and differentiated needs to be further elucidated, and there is evidence that the tumour microenvironment governs cancer stemness. Using PLR123, a colon cancer cell line with CSC properties, we determined the culture conditions necessary to establish a pair of three-dimensional (3D) culture models grown in Matrigel, designated stemCO and diffCO. The conditions were determined by comparing the phenotypes in the models with PLR123 mouse xenografts colonising lung and liver. StemCO resembled LGR5-positive undifferentiated tumours in the lung, and diffCO had lumen structures composed of polarised cells that were similar to the ductal structures found in differentiated tumours in the liver. In a case using the models for biomedical research, treatment with JAG-1 peptide or a γ-secretase inhibitor modified the Notch signaling and induced changes indicating that the signal participates in lumen formation in the models. Our results demonstrate that culture conditions affect the stemness of 3D culture models generated from CSCs and show that comparing models with different phenotypes is useful for studying how the tumour environment regulates cancer.
Project description:Radiation therapy is one of the most effective tools in cancer therapy. However, success varies individually, necessitating improved understanding of radiobiology. Three-dimensional (3D) tumor spheroids are increasingly gaining attention, being a superior in vitro cancer model compared to 2D cell cultures. This in vitro study aimed at comparing radiation responses in 2D and 3D cell culture models of different human cancer cell lines (PC-3, LNCaP and T-47D) irradiated with varying doses (1, 2, 4, 6, 8 or 20 Gy) of X-ray beams. Radiation response was analyzed by growth analysis, various cell viability assays (e.g., clonogenic assay, resazurin assay) and amount of DNA damage (γH2AX Western Blot). Results showed decreasing cell proliferation with the increase of radiation doses for all cell lines in monolayers and spheroids of LNCaP and T-47D. However, significantly lower radiosensitivity was detected in spheroids, most pronounced in PC-3, evincing radiation resistance of PC-3 spheroids up to 8 Gy and significant growth inhibition only by a dose escalation of 20 Gy. Cell line comparison showed highest radiosensitivity in LNCaP, followed by T-47D and PC-3 in 2D, whereas, in 3D, T-47D showed highest sensitivity. The results substantiate the significant differences in radiobiological response to X-rays between 2D and 3D cell culture models.
Project description:Cultured cell lines are widely used for research in the physiology, pathophysiology, toxicology and pharmacology of the renal proximal tubule. The lines that are most appropriate for a given use depend on the genes expressed. New tools for transcriptomic and proteomic profiling using RNA-sequencing (RNA-Seq) and mass spectrometry make it possible to catalog expressed genes in each cell line. This data set is the protoemic data of Rat NRK-52E cell line. We concludeno cell line fully matched the transcriptome of native proximal tubule cells. However, some of the lines tested are suitable for the study of particular metabolic and transport processes seen in the proximal tubule.
Project description:The hypothesis that the onset and development of colorectal cancer result from the alteration of the close cooperation of mRNA-miRNA has gained increasing popularity. We performed a multifaceted enrichment analysis of hypernetworks, taking advantage of the simultaneous evaluation of transcriptome and miRNAome, to search deregulated micro-societies of mRNAs and miRNAs involved in signaling pathways that become critical when altered in colorectal carcinogenesis.
Project description:Two-dimensional cell cultures are established models in research for studying and perturbing cell-type specific functions. However, many limitations apply to the cell growth in a monolayer using standard cell culture media. Although they have been used for decades, their formulations do not mimic the composition of the human cell environment. In this study, we analyzed the impact of a newly formulated human plasma-like media (HPLM) on cell proliferation, mitochondrial bioenergetics, and alterations of drug efficacies using three distinct cancer cell lines. Using high-resolution respirometry, we observed that cells grown in HPLM displayed significantly altered mitochondrial bioenergetic profiles, particularly related to mitochondrial density and mild uncoupling of respiration. Furthermore, in contrast to standard media, the growth of cells in HPLM unveiled mitochondrial dysfunction upon exposure to the FDA-approved kinase inhibitor sunitinib. This seemingly context-dependent side effect of this drug highlights that the selection of the cell culture medium influences the assessment of cancer drug sensitivities. Thus, we suggest to prioritize media with a more physiological composition for analyzing bioenergetic profiles and to take it into account for assigning drug efficacies in the cell culture model of choice.
Project description:How to efficiently obtain high-purity cancer stem cells (CSCs) has been the basis of CSC research, but the optimal conditions for serum-free suspension culture of CSCs are still unclear. The present study aimed to define the optimal culture medium composition and culture time for the enrichment of colon CSCs via suspension culture. Suspension cell cultures of colon cancer DLD-1 cells were prepared using serum-free medium (SFM) containing variable concentrations of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) to produce spheroids. Culture times were set at 10, 20 and 30 days. A total of nine different concentrations of EGF and bFGF were added to SFM to generate nine experimental groups. The proportions of CD44+, CD133+, and CD44+CD133+ double-positive spheroid cells were detected via flow cytometry. mRNA expression of stemness-, epithelial-mesenchymal transition- and Wnt/β-catenin pathway-associated genes was determined via reverse transcription-quantitative PCR. Self-renewal ability was evaluated by a sphere-forming assay. Tumorigenesis was studied in vitro using a colony formation assay and in vivo via subcutaneous cell injection in nude mice. It was found that the highest expression proportions of CD133+ and CD44+ spheroid cells were observed in group (G)9 (20 ng/ml EGF + 20 ng/ml bFGF) at 30 days (F=123.554 and 99.528, respectively, P<0.001), CD133+CD44+ cells were also observed in G9 at 30 days (and at 10 days in G3 and 20 days in G6; F=57.897, P<0.001). G9 at 30 days also displayed the highest expression of Krüppel-like factor 4, leucine-rich repeat-containing G protein-coupled receptor 5, CD44, CD133, Vimentin and Wnt-3a (F=22.682, 25.401, 3.272, 7.852, 13.331 and 17.445, respectively, P<0.001) and the lowest expression of E-cadherin (F=10.851, P<0.001). G9 at 30 days produced the highest yield of cell spheroids, as determined by a sphere forming assay (F=19.147, P<0.001); colony formation assays also exhibited the greatest number of colonies derived from G9 spheroids at 30 days (F=60.767, P<0.01), which also generated the largest mean tumor volume in the subcutaneous tumorigenesis xenograft model (F=12.539, P<0.01). In conclusion, 20 ng/ml EGF + 20 ng/ml bFGF effectively enriched colon CSCs when added to suspension culture for 30 days, and conferred the highest efficiency compared with other combinations.
Project description:Ovine mastitis is defined as the inflammation of the sheep udder, most commonly caused in response to intramammary infections. Based on the occurrence of clinical signs, mastitis is characterized as either clinical or subclinical (SCM). The impact of ovine SCM on the overall sustainability of dairy sheep farms has been documented underpinning the significance of efficient diagnosis. Although SCM can be detected in cows, the performance and the validity of the methods used do not transfer in dairy sheep. This fact challenges the development of evidence-based ovine udder health management protocols and renders the detection and control of ovine mastitis rather problematic. Currently, cell culture-based models are being successfully used in biomedical studies and have also been effectively used in the case of bovine mastitis. The objective of the present study was to culture ovine primary mammary cells for the development of 2D and 3D cell culture-based models for the study of ovine SCM. Cells were infected by mastitis-inducing pathogens mimicking the pathogenesis of SCM as derived by natural intramammary infections. The secreted proteins were subjected to mass-spectrometry resulting in the identification of 79 distinct proteins. Among those, several had already been identified in healthy or mastitic milk, while others were detected for the first time in the ovine mammary secretome. The development of cell-based models for the early detection and the overall study of SCM has the potential to be applicable and beneficial for the udder health management in dairy sheep.