Capturing complex tumour biology in vitro: histological and molecular characterisation of precision cut slices.
Ontology highlight
ABSTRACT:
SUBMITTER: Davies EJ
PROVIDER: S-EPMC4673528 | biostudies-literature | 2015 Dec
REPOSITORIES: biostudies-literature
Publications
Capturing complex tumour biology in vitro: histological and molecular characterisation of precision cut slices.
Davies Emma J EJ Dong Meng M Gutekunst Matthias M Närhi Katja K van Zoggel Hanneke J A A HJ Blom Sami S Nagaraj Ashwini A Metsalu Tauno T Oswald Eva E Erkens-Schulze Sigrun S Delgado San Martin Juan A JA Turkki Riku R Wedge Stephen R SR af Hällström Taija M TM Schueler Julia J van Weerden Wytske M WM Verschuren Emmy W EW Barry Simon T ST van der Kuip Heiko H Hickman John A JA
Scientific reports 20151209
Precision-cut slices of in vivo tumours permit interrogation in vitro of heterogeneous cells from solid tumours together with their native microenvironment. They offer a low throughput but high content in vitro experimental platform. Using mouse models as surrogates for three common human solid tumours, we describe a standardised workflow for systematic comparison of tumour slice cultivation methods and a tissue microarray-based method to archive them. Cultivated slices were compared to their in ...[more]
Similar Datasets
Project description:To assess chemical toxicity, current high throughput screening (HTS) assays rely primarily on in vitro measurements using cultured cells. Responses frequently differ from in vivo results due to the lack of physical and humoral interactions provided by the extracellular matrix, cell-cell interactions, and other molecular components of the native organ. To more accurately reproduce organ complexity in HTS, we developed an organotypic assay using the cryopreserved precision cut lung slice (PCLS) from rats and mice. Compared to the never-frozen PCLS, their frozen-thawed counterpart slices showed viability or metabolic activity that is decreased to an extent comparable to that observed in other cryopreserved cells and tissues, but shows no differences in further changes in cell viability, mitochondrial integrity, and glutathione activity in response to the model toxin zinc chloride (ZnCl2). Notably, these measurements were successfully miniaturized so as to establish HTS capacity in a 96-well plate format. Finally, PCLS responses correlated with common markers of lung injury measured in lavage fluid from rats intratracheally instilled with ZnCl2. In summary, we establish that the cryopreserved PCLS is a feasible approach for HTS investigations in predictive toxicology.
| S-EPMC5009619 | biostudies-literature
Project description:Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly, chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.
| S-EPMC5512285 | biostudies-literature
Project description:BackgroundRespiratory diseases represent a global health burden. Because research on therapeutic strategies of airway diseases is essential, the technique of precision-cut lung slices (PCLS) has been developed and widely studied. PCLS are an alternative ex vivo model and have the potential to replace and reduce in vivo animal models. So far, the majority of studies was conducted with short-term cultivated PCLS (≤ 72 h). As there is large interest in research of chronic diseases and chronic toxicity, feasibility of cultivating human PCLS long-term over 2 weeks and recently over 4 weeks was investigated by another research group with successful results. Our aim was to establish a model of long-term cultivated rat PCLS over a period of 29 days.MethodsRat PCLS were cultured for 29 days and analysed regarding viability, histopathology, reactivity and gene expression at different time points during cultivation.ResultsCultivation of rat PCLS over a 29-day time period was successful with sustained viability. Furthermore, the ability of bronchoconstriction was maintained between 13 and 25 days, depending on the mediator. However, reduced relaxation, altered sensitivity and increased respiratory tone were observed. Regarding transcription, alteration in gene expression pattern of the investigated target genes was ascertained during long-term cultivation with mixed results. Furthermore, the preparation of PCLS seems to influence messenger ribonucleic acid (mRNA) expression of most target genes. Moreover, the addition of fetal bovine serum (FBS) to the culture medium did not improve viability of PCLS. In contrast to medium without FBS, FBS seems to affect measurements and resulted in marked cellular changes of metaplastic and/or regenerative origin.ConclusionsOverall, a model of long-term cultivated rat PCLS which stays viable for 29 days and reactive for at least 13 days could be established. Before long-term cultivated PCLS can be used for in-depth study of chronic diseases and chronic toxicity, further investigations have to be made.
| S-EPMC9490993 | biostudies-literature
Project description:Chronic obstructive pulmonary disease (COPD) is the third leading cause of death globally and constitutes a major health problem. The disease is characterized by airflow obstructions due to chronic bronchitis and/or emphysema. Emerging evidence suggests that COPD is the result of impaired epithelial repair. Motivated by the need for more effective treatments, we studied whether receptor activator of nuclear factor κ-Β ligand (RANKL) contributed to epithelial repair, as this protein has been implicated in epithelial regeneration of breast and thymus. To do so, we used precision-cut lung slices prepared from mouse tissue-viable explants that can be cultured ex vivo for up to a few days while retaining features of lung tissue. Slices were cultured with 10, 100, or 500 ng/ml of mouse RANKL for 24 h. We first found RANKL activated nuclear factor κ-Β signaling, which is involved in cellular stress responses, without affecting the general viability of slices. Cell proliferation, however, was not altered by RANKL treatment. Interestingly, RANKL did reduce cell death, as revealed by TUNEL stainings and profiling of apoptosis-related proteins, indicating that it contributes to repair by conferring protection against cell death. This study improves our understanding of lung repair and could create new opportunities for developing COPD treatments.
| S-EPMC9659591 | biostudies-literature
Project description:Two important signaling pathways in liver fibrosis are the PDGF- and TGF? pathway and compounds inhibiting these pathways are currently developed as antifibrotic drugs. Testing antifibrotic drugs requires large numbers of animal experiments with high discomfort. Therefore, a method to study these drugs ex vivo was developed using precision-cut liver slices from fibrotic rat livers (fPCLS), representing an ex vivo model with a multicellular fibrotic environment. We characterized the fibrotic process in fPCLS from rat livers after 3 weeks of bile duct ligation (BDL) during incubation and tested compounds predominantly inhibiting the TGF? pathway (perindopril, valproic acid, rosmarinic acid, tetrandrine and pirfenidone) and PDGF pathway (imatinib, sorafenib and sunitinib). Gene expression of heat shock protein 47 (Hsp47), ? smooth muscle actin (?Sma) and pro-collagen 1A1 (Pcol1A1) and protein expression of collagens were determined. During 48 hours of incubation, the fibrosis process continued in control fPCLS as judged by the increased gene expression of the three fibrosis markers, and the protein expression of collagen 1, mature fibrillar collagen and total collagen. Most PDGF-inhibitors and TGF?-inhibitors significantly inhibited the increase in gene expression of Hsp47, ?Sma and Pcol1A1. Protein expression of collagen 1 was significantly reduced by all PDGF-inhibitors and TGF?-inhibitors, while total collagen was decreased by rosmarinic acid and tetrandrine only. However, fibrillar collagen expression was not changed by any of the drugs. In conclusion, rat fPCLS can be used as a functional ex vivo model of established liver fibrosis to test antifibrotic compounds inhibiting the PDGF- and TGF? signalling pathway.
| S-EPMC3995767 | biostudies-literature
Project description:BackgroundAsthma exacerbations evoke emergency room visits, progressive loss of lung function and increased mortality. Environmental and industrial toxicants exacerbate asthma, although the underlying mechanisms are unknown. We assessed whether 3 distinct toxicants, salicylic acid (SA), toluene diisocyanate (TDI), and 1-chloro-2,4-dinitrobenzene (DNCB) induced airway hyperresponsiveness (AHR) through modulating excitation-contraction coupling in human airway smooth muscle (HASM) cells. The toxicants include a non-sensitizing irritant (SA), respiratory sensitizer (TDI) and dermal sensitizer (DNCB), respectively. We hypothesized that these toxicants induce AHR by modulating excitation-contraction (EC) coupling in airway smooth muscle (ASM) cells.MethodsCarbachol-induced bronchoconstriction was measured in precision-cut human lung slices (hPCLS) following exposure to SA, TDI, DNCB or vehicle. Culture supernatants of hPCLS were screened for mediator release. In HASM cells treated with the toxicants, surrogate readouts of EC coupling were measured by phosphorylated myosin light chain (pMLC) and agonist-induced Ca2+ mobilization ([Ca2+]i). In addition, Nrf-2-dependent antioxidant response was determined by NAD(P) H quinone oxidoreductase 1 (NQO1) expression in HASM cells.ResultsIn hPCLS, SA, but not TDI or DNCB, potentiated carbachol-induced bronchoconstriction. The toxicants had little effect on release of inflammatory mediators, including IL-6, IL-8 and eotaxin from hPCLS. In HASM cells, TDI amplified carbachol-induced MLC phosphorylation. The toxicants also had little effect on agonist-induced [Ca2+]i. CONCLUSION: SA, a non-sensitizing irritant, amplifies agonist-induced bronchoconstriction in hPCLS via mechanisms independent of inflammation and Ca2+ homeostasis in HASM cells. The sensitizers TDI and DNCB, had little effect on bronchoconstriction or inflammatory mediator release in hPCLS.ImplicationsOur findings suggest that non-sensitizing irritant salicylic acid may evoke AHR and exacerbate symptoms in susceptible individuals or in those with underlying lung disease.
| S-EPMC6458705 | biostudies-literature
Project description:Organotypic lung slices, sometimes known as precision-cut lung slices (PCLS), provide an environment in which numerous cell types and interactions can be maintained outside the body (ex vivo). PCLS were maintained ex vivo for up to a week and demonstrated health via the presence of neurons, maintenance of tissue morphology, synthesis of mucopolysaccharides, and minimal cell death. Multiple phenotypes of neuronal fibers were present in lung slices with varied size, caliber, and neurotransmitter immunoreactivity. Of the neuropeptides present in fibers, calcitonin gene-related peptide (CGRP) was the most prevalent. Exposing PCLS to recombinant CGRP resulted in the proliferation and dispersion of CD19+ B cells in slices taken selectively from females. The number of granules containing immunoreactive (ir) surfactant protein C (SPC), which are representative of alveolar type 2 cells, increased in slices from females within 24 h of exposure to CGRP. Additionally, ir-SPC granule size increased in slices from males and females across 48 h of exposure to CGRP. Exposure of PCLS to exogenous CGRP did not alter the number of solitary pulmonary neuroendocrine cells (PNEC) but did result in neuroendocrine bodies that had significantly more cells. Neuronal fiber numbers were unchanged based on ir-peripherin; however, ir-CGRP became non-detectable in fibers while unchanged in PNECs. The effects of exogenous CGRP provide insight into innate immune and neuroendocrine responses in the lungs that may be partially regulated by neural fibers. The sex-dependent nature of these changes may point to the basis for sex-selective outcomes among respiratory diseases.
| S-EPMC10665790 | biostudies-literature
Project description:Human precision-cut lung slices (PCLS) have proven to be an invaluable tool for numerous toxicologic, pharmacologic, and immunologic studies. Although a cultivation period of <1 week is sufficient for most studies, modeling of complex disease mechanisms and investigating effects of long-term exposure to certain substances require cultivation periods that are much longer. So far, data regarding tissue integrity of long-term cultivated PCLS are incomplete. More than 1500 human PCLS from 16 different donors were cultivated under standardized, serum-free conditions for up to 28 days and the viability, tissue integrity, and the transcriptome was assessed in great detail. Even though viability of PCLS was well preserved during long-term cultivation, a continuous loss of cells was observed. Although the bronchial epithelium was well preserved throughout cultivation, the alveolar integrity was preserved for about 2 weeks, and the vasculatory system experienced significant loss of integrity within the first week. Furthermore, ciliary beat in the small airways gradually decreased after 1 week. Interestingly, keratinizing squamous metaplasia of the alveolar epithelium with significantly increasing manifestation were found over time. Transcriptome analysis revealed a significantly increased immune response and significantly decreased metabolic activity within the first 24 hours after PCLS generation. Overall, this study provides a comprehensive overview of histomorphologic and pathologic changes during long-term cultivation of PCLS.
| S-EPMC8891143 | biostudies-literature
Project description:BACKGROUND:Intestinal fibrosis is a hallmark of Crohn's disease. Here, we investigated the impact of several putative antifibrotic compounds on the expression of fibrosis markers using murine precision-cut intestinal slices. METHODS:Murine precision-cut intestinal slices were cultured for 48 hours in the presence of profibrotic and/or antifibrotic compounds. The fibrotic process was studied on gene and protein level using procollagen 1a1 (Col1?1), heat shock protein 47 (Hsp47), fibronectin (Fn2), and plasminogen activator inhibitor-1 (Pai-1). The effects of potential antifibrotic drugs mainly inhibiting the transforming growth factor ? (TGF-?) pathway (eg, valproic acid, tetrandrine, pirfenidone, SB203580, and LY2109761) and compounds mainly acting on the platelet-derived growth factor (PDGF) pathway (eg, imatinib, sorafenib, and sunitinib) were assessed in the model at nontoxic concentrations. RESULTS:Murine precision-cut intestinal slices remained viable for 48 hours, and an increased expression of fibrosis markers was observed during culture, including Hsp47, Fn2, and Pai-1. Furthermore, TGF-?1 stimulated fibrogenesis, whereas PDGF did not have an effect. Regarding the tested antifibrotics, pirfenidone, LY2109761, and sunitinib had the most pronounced impact on the expression of fibrosis markers, both in the absence and presence of profibrotic factors, as illustrated by reduced levels of Col1?1, Hsp47, Fn2, and Pai-1 after treatment. Moreover, sunitinib significantly reduced Hsp47 and Fn2 protein expression and the excretion of procollagen 1. CONCLUSIONS:Precision-cut intestinal slices can successfully be used as a potential preclinical screening tool for antifibrotic drugs. We demonstrated that sunitinib reduced the expression of several fibrosis markers, warranting further evaluation of this compound for the treatment of intestinal fibrosis.
| S-EPMC7150673 | biostudies-literature
Project description:BackgroundMonitoring cellular processes across different levels of complexity, from the cellular to the tissue scale, is important for understanding tissue structure and function. However, it is challenging to monitor and estimate these structural and dynamic interactions within three-dimensional (3D) tissue models.ObjectiveThe aim of this study was to design a method for imaging, tracking, and quantifying 3D changes in cell morphology (shape and size) within liver tissue, specifically a precision-cut liver slice (PCLS). A PCLS is a 3D model of the liver that allows the study of the structure and function of liver cells in their native microenvironment.MethodsHere, we present a method for imaging liver tissue during anisosmotic exposure in a multispectral four-dimensional manner. Three metrics of tissue morphology were measured to quantify the effects of osmotic stress on liver tissue. We estimated the changes in the volume of whole precision cut liver slices, quantified the changes in nuclei position, and calculated the changes in volumetric responses of tissue-embedded cells.ResultsDuring equilibration with cell-membrane-permeating and non-permeating solutes, the whole tissue experiences shrinkage and expansion. As nuclei showed a change in position and directional displacement under osmotic stress, we demonstrate that nuclei could be used as a probe to measure local osmotic and mechanical stress. Moreover, we demonstrate that cells change their volume within tissue slices as a result of osmotic perturbation and that this change in volume is dependent on the position of the cell within the tissue and the duration of the exposure.ConclusionThe results of this study have implications for a better understanding of multiscale transport, mechanobiology, and triggered biological responses within complex biological structures.
| S-EPMC10903333 | biostudies-literature