Compressed collagen and decellularized tissue: novel components in a pipeline approach for the study of cancer metastasis

Metastasis is a complex process which is difficult to study and model. Experimental ingenuity is therefore essential when seeking to elucidate the biological mechanisms involved. Typically, in vitro models of metastasis have been overly simplistic, lacking the characteristic elements of the tumour microenvironment, whereas in vivo models are expensive, requiring specialist resources. Here we propose a pipeline approach for the study of cell migration and colonization, two critical steps in the metastatic cascade.We used a range of extracellular matrix derived contexts to facilitate a progressive approach to the observation and quantification of cell behaviour in 2D, 3D and at border zones between dimensions. At the simplest level, cells were set onto collagen-coated plastic or encapsulated within a collagen matrix. To enhance this, a collagen compression technique provided a stiffened, denser substrate which could be used as a 2D surface or to encapsulate cells. Decellularized tissue from the chorioallantoic membrane of the developing chicken embryo was used to provide a more structured, biologically relevant extracellular matrix-based context in which cell behaviour could then be compared with its in vivo counterpart.Cell behaviour could be observed and quantified within each context using standard laboratory techniques of microscopy and immunostaining, affording the opportunity for comparison and contrast of behaviour across the whole range of contexts. In particular, the temporal constraints of the in vivo CAM were removed when cells were cultured on the decellularized CAM, allowing for much longer-term cell colonization and cell-cell interaction.Together the assays within this pipeline provide the opportunity for the study of cell behaviour in a replicable way across multiple environments. The assays can be set up and analysed using easily available resources and standard laboratory equipment. We believe this offers the potential for the detailed study of cell migration and colonization of tissue, essential steps in the metastatic cascade. Also, we propose that the pipeline could be used in the wider arena of cell culture in general with the increasingly more complex contexts allowing cell behaviours and interactions to be explored in a stepwise fashion in an integrated way

3D extracellular matrix microenvironment in bioengineered tissue models of primary pediatric and adult brain tumors.

Dynamic alterations in the unique brain extracellular matrix (ECM) are involved in malignant brain tumors. Yet studies of brain ECM roles in tumor cell behavior have been difficult due to lack of access to the human brain. We present a tunable 3D bioengineered brain tissue platform by integrating microenvironmental cues of native brain-derived ECMs and live imaging to systematically evaluate patient-derived brain tumor responses. Using pediatric ependymoma and adult glioblastoma as examples, the 3D brain ECM-containing microenvironment with a balance of cell-cell and cell-matrix interactions supports distinctive phenotypes associated with tumor type-specific and ECM-dependent patterns in the tumor cells\u27 transcriptomic and release profiles. Label-free metabolic imaging of the composite model structure identifies metabolically distinct sub-populations within a tumor type and captures extracellular lipid-containing droplets with potential implications in drug response. The versatile bioengineered 3D tumor tissue system sets the stage for mechanistic studies deciphering microenvironmental role in brain tumor progression

Chromosome engineering in mice

CHROMOSOMAL rearrangements are the major cause of inherited human disease and fetal loss 1 . Translations 2 and loss of heterozygosity 3 are important genetic changes causally involved in neoplasia. Chromosomal variants, such as deficiencies, are commonly exploited in genetic screens in organisms such as Drosophila because a small portion of the genome is functionally hemizygous 4 . In the mouse, deficiencies are not generally available, thus genetic screens for recessive mutations are cumbersome 5 . We report here that defined deficiencies, inversions and duplications extending to 3-4 cM can be constructed in embryonic stem cells. This was achieved by consecutive targeting of loxP recombination substrates to the end points of a genetic interval followed by Cre-induced recombination. This reconstructs a positive selectable marker which facilitates direct selection of clones with a chromosome structure specific to the relative orientation of the loxP sites. Duplication and deletion alleles have been transmitted into the mouse germ line. The availability of mice with defined regions of segmental haploidy will allow their use in genetic screens and enable accurate models of human 'chromosomal' diseases to be generated. © 1995 Nature Publishing Group.Link_to_subscribed_fulltex