Cancer Biology

Despite their involvement in many physiological and pathological processes, fibroblasts remain a poorly-characterized cell type. Analysis of primary fibroblasts while maintaining their in vivo phenotype is challenging: standard methods for fibroblast isolation require cell culture in vitro, which is known to alter phenotypes. Previously-described protocols for the dissociation of primary tissues fail to extract sufficient numbers of fibroblasts, instead largely yielding immune cells. Here, we describe an optimized method for generating a fibroblast-enriched single-cell suspension from human tissues using combined mechanical and enzymatic dissociation. This allows analysis of ex vivo fibroblasts without the need for culture in vitro.

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.

The three-dimensional organisation of cells in a tissue and their interaction with adjacent cells and extracellular matrix is a key determinant of cellular responses, including how tumour cells respond to stress conditions or therapeutic drugs (Elliott and Yuan, 2011). In vivo, tumour cells are embedded in a stroma formed primarily by fibroblasts that produce an extracellular matrix and enwoven with blood vessels. The 3D mixed cell type spheroid model described here incorporates these key features of the tissue microenvironment that in vivo tumours exist in; namely the three-dimensional organisation, the most abundant stromal cell types (fibroblasts and endothelial cells), and extracellular matrix. This method combined with confocal microscopy can be a powerful tool to carry out drug sensitivity, angiogenesis and cell migration/invasion assays of different tumour types.

Galectin-3 is a member of a class of proteins termed Galectins, characterized by their ability to bind glycans containing β-galactose (Cummings and Liu, 2009). Galectin-3 binds preferentially to proteoglycans terminating with N-acetyllactosamine (LacNAc) chains (i.e., tandem repeats of galactose) (Newlaczyl and Yu, 2011). Galectin-3 is unique among the galectins in its chimeric structure. It shares a conserved carbohydrate recognition domain (CRD) with the other galectins, but has a long amino-terminal tail that is thought to be involved in protein aggregation. It can also form homodimers through its CRD (Cummings and Liu, 2009). Galectin-3 has been found to have diverse functions in tumorigenesis including: signaling, apoptosis inhibition, immune suppression, cell growth, and metastasis among others. Galectin-3 is frequently upregulated in cancers (Nangia-Makker et al., 2008). Its function largely depends on its expression and localization properties (Newlaczyl and Yu, 2011). Because of its many roles in cancer-associated processes, establishing a method for Galectin-3 production is valuable for further study of its functions in cancer. Here, we describe how Galectin-3 purification was achieved by cloning of the human Galectin-3 gene into pGEX-2T vector containing the gene for glutathione-S-transferase (GST) upstream of its cloning site. The Galectin-3 gene was cloned into this vector via restriction digests of both the plasmid and the Galectin-3 gene by restriction enzymes BamHI and EcoRI, followed by ligation of the two fragments. The resulting plasmid was then used to transform BL21, an Escherichia coli (E. coli) strain specialized for protein expression. Finally, we discuss how the GST fusion protein was isolated and the recombinant Galectin-3 protein was further purified from the GST.

The assay was developed to investigate the impact of stromal cells of different types (in our case breast cancer associated fibroblasts stably manipulated to modify expression of genes of interest) on the invasive capacity of epithelial cancer cells (in our case breast cancer cell lines) (Verghese et al., 2013). Typical two dimensional invasion assays do necessarily account for the presence of extracellular matrix that is present around the stromal and tumour cells in vivo and therefore cellular behaviour within these cultures may be non-physiological. This spheroid assay was developed to attempt to replicate more closely the environment that is present around breast cancer stromal and tumour cells in actual tumours (Verghese et al., 2013). Extra cellular matrix composed of both collagen IV and collagen I is included and fibroblasts and epithelial cells are given the opportunity to develop “physiological” interactions (Verghese et al., 2013; Hooper et al., 2006). The method was developed from Nowicki et al. (2008), and we have published data using it in Verghese et al. (2013).

The invasive ability of cancer cells is a crucial function for cancer metastasis and the surrounding microenvironment of cancer cells in living tissues is three-dimension (3D). Therefore, to establish an in vitro invasion assay in a 3D system to predict cancer invasive ability is valuable in the research for cancer metastasis. Here, we describe a 3D invasion assay for observing the morphology and comparing the invasive ability of cancer cells in artificial 3D environments (Yang et al., 2012). Collagen I gels are used to cover on the top of cancer cells attached on coverslip glass dish and medium containing FBS is added as a chemoattractant. After incubation for a suitable time, the cells are fixed and stained. The invasion index can be calculated and the morphology can be imaged with a laser confocal microscope.