Cancer Biology


Protocols in Current Issue
Protocols in Past Issues
0 Q&A 1253 Views Dec 20, 2022

The extracellular matrix (ECM) is a non-cellular network of macromolecules, which provides cells and tissues with structural support and biomechanical feedback to regulate cellular function, tissue tension, and homeostasis. Even subtle changes to ECM abundance, architecture, and organization can affect downstream biological pathways, thereby influencing normal cell and tissue function and also driving disease conditions. For example, in cancer, the ECM is well known to provide both biophysical and biochemical cues that influence cancer initiation, progression, and metastasis, highlighting the need to better understand cell–ECM interactions in cancer and other ECM-enriched diseases. Initial cell-derived matrix (CDM) models were used as an in vitro system to mimic and assess the physiologically relevant three-dimensional (3D) cell–ECM interactions. Here, we describe an expansion to these initial CDM models generated by fibroblasts to assess the effect of genetic or pharmacological intervention on fibroblast-mediated matrix production and organization. Additionally, we highlight current methodologies to quantify changes in the ultrastructure and isotropy of the resulting ECM and also provide protocols for assessing cancer cell interaction with CDMs. Understanding the nature and influence of these complex and heterogeneous processes can offer insights into the biomechanical and biochemical mechanisms, which drive cancer development and metastasis, and how we can target them to improve cancer outcomes.

0 Q&A 4338 Views Jan 20, 2021

Research on cell migration and interactions with the extracellular matrix (ECM) was mostly focused on 2D surfaces in the past. Many recent studies have highlighted differences in migratory behaviour of cells on 2D surfaces compared to complex cell migration modes in 3D environments. When embedded in 3D matrices, cells constantly sense the physicochemical, topological and mechanical properties of the ECM and adjust their behaviour accordingly. Changes in the stiffness of the ECM can have effects on cell morphology, differentiation and behaviour and cells can follow stiffness gradients in a process called durotaxis. Here we introduce a detailed protocol for the assembly of 3D matrices consisting of collagen I/fibronectin and embedding cells for live cell imaging. Further, we will show how the matrix can be stiffened via non-enzymatic glycation and how collagen staining with fluorescent dyes allows simultaneous imaging of both matrix and cells. This approach can be used to image cell migration in 3D microenvironments with varying stiffness, define cell-matrix interactions and the cellular response to changing ECM, and visualize matrix deformation by the cells.

0 Q&A 2768 Views Nov 20, 2020
A clear understanding of nanoparticle interactions with living systems at the cellular level is necessary for developing nanoparticle-based therapeutics. Magnetic iron oxide nanoparticles provide unique opportunities to study these interactions because of their responsiveness to magnetic fields. This enables sorting of cells containing nanoparticles from in vivo models. Once sorted, flow cytometry can identify individual cell types, which can be further analyzed for iron content, gene or protein expression changes associated with nanoparticle uptake, and for other biological responses at a molecular level. Here we provide a detailed protocol to sort and identify cells in the tumor microenvironment that have internalized magnetic iron oxide nanoparticles following intravenous administration.
0 Q&A 4300 Views May 20, 2020
Differential exposure of tumor cells to microenvironmental cues greatly impacts cell phenotypes, raising a need for position based sorting of tumor cells amenable to multiple OMICs and functional analyses. One such key determinant of tumor heterogeneity in solid tumors is its vasculature. Proximity to blood vessels (BVs) profoundly affects tumor cell phenotypes due to differential availability of oxygen, gradient exposure to blood-borne substances and inputs by angiocrine factors. To unravel the whole spectrum of genes, pathways and phenotypes impacted by BVs and to determine spatial domains of vascular influences, we developed a methodology for sorting tumor cells according to their relative distance from BVs. The procedure exemplified here using glioblastoma (GBM) model is based on differential uptake of intra-venously injected, freely-diffusing fluorescent dye that allows separation of stroma-free tumor cells residing in different, successive microenvironments amenable for subsequent OMICs and functional analyses. This reliable, easy to use, cost effective strategy can be extended to all solid tumors to study the impact of vasculature or the lack of it.
0 Q&A 5674 Views Sep 20, 2019
Dissolved oxygen and its availability to cells in culture is an overlooked variable which can have significant consequences on experimental research outcomes, including reproducibility. Oxygen sensing pathways play key roles in cell growth and behavior and pericellular oxygen levels should be controlled when establishing in vitro models. Standard cell culture techniques do not have adequate control over pericellular oxygen levels. Slow diffusion through culture media limits the precision of oxygen delivery to cells, making it difficult to accurately reproduce in vivo-like oxygen conditions. Furthermore, different types of cells consume oxygen at varying rates and this can be affected by the density of growing cells. Here, we describe a novel in vitro system that utilizes hypoxic chambers and oxygen-permeable culture dishes to control pericellular oxygen levels and provide rapid oxygen delivery to adherent cells. This procedure is particularly relevant for protocols studying effects of rapid oxygen changes or intermittent hypoxia on cellular behavior. The system is inexpensive and easily assembled without highly specialized equipment.

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