Abstract
The metastatic spread of cancer cells is a step-wise process that starts with dissociation from primary tumours and local invasion of adjacent tissues. The ability to invade local tissues is the product of several processes, including degradation of extracellular matrices (ECM) and movement of tumour cells through physically-restricting gaps. To identify properties contributing to tumour cells squeezing through narrow gaps, invasive MDA-MB-231 human breast cancer and MDA-MB-435 human melanoma cells were subjected to three successive rounds of selection using cell culture inserts with highly constraining 3 μm pores. For comparison purposes, flow cytometry was also employed to enrich for small diameter MDA-MB-231 cells. RNA-Sequencing (RNA-seq) using the Illumina NextSeq 500 platform was undertaken to characterize how gene expression differed between parental, invasive pore selected or small diameter cells. Gene expression results obtained by RNA-seq were validated by comparing with RT-qPCR. Transcriptomic data generated could be used to determine how alterations that enable cell passage through narrow spaces contribute to local invasion and metastasis.
Highlights
The metastatic spread of cancer cells from primary tumours to distant sites is the most serious and deadliest aspect of the disease, with estimates of up to 90% of cancer related deaths being directly associated with metastasis[1,2]
It has become apparent that the same processes that contribute to cancer metastasis facilitate primary tumour growth[3,4]
The movement away from the tumour into adjacent tissue may be promoted by changes in migratory behaviour, increased extracellular matrix (ECM) degrading activity, and the ability to squeeze between cells and ECM protein fibres[7]
Summary
The metastatic spread of cancer cells from primary tumours to distant sites is the most serious and deadliest aspect of the disease, with estimates of up to 90% of cancer related deaths being directly associated with metastasis[1,2]. As a result, characterizing the properties of metastatic cells that enable their dissemination may identify actionable targets for chemotherapy that could reduce cancer spread and possibly tumour growth and progression. It can be predicted that changes in the deformability of tumour cells that enabled their movement through physically constraining conditions in their three dimensional environment would likely promote cancer spread[8]. To determine how gene expression differs between Parental, Selected and FlowSorted populations of MDA-MB-231 and MDA-MB-435, total RNA was extracted and enriched for polyA+ mRNAs, and subjected to RNA-Sequencing (Fig. 2 and Table 1). The transcriptomic data generated by this study may reveal important modifiers of the physical properties that enable tumour cells to move through narrow spaces, as well as regulators of cell size, that contribute to the metastatic spread of cancer
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