Background

Tumor invasion refers to the process by which cancer cells penetrate the basement membrane into surrounding stroma, neighboring blood vessels, and lymph nodes (Stuelten et al., 2018; Baghban et al., 2020). Tumor invasion is an essential step for metastasis of human cancers (Baghban et al., 2020). Amongst the many steps of metastasis, the process of tumor invasion has been targeted by many therapeutic agents to suppress the distant spread of tumors (Gandalovicova et al., 2017). The Boyden chamber assay is the benchmark technique for measuring the invasion of cancer cells (Guy et al., 2017). This assay involves the movement of a single-cell suspension through a microscopic mesh (8 micrometers in pore size) coated with an extracellular matrix. However, this assay suffers from several caveats. The physiological relevance of transwell inserts (to recapitulate invasion in vivo) is poor. The pore size of the membrane highly influences the number of cells that invade (Guy et al., 2017). In the Boyden chamber assay, a chemokine is added to the lower basolateral chamber to facilitate the invasion of tumor cells through the transwell membrane; however, it is very difficult to control the concentration of the chemokine gradient, and this may produce aberrant results (Guy et al., 2017). The Boyden chamber is one of the prevalent methods to measure tumor cell invasion. The “wound-healing assay” measures the migration (NOT THE INVASION) of human cancer cells. There are subtle differences between a “migration assay” and an “invasion assay”(Justus et al., 2014; Pijuan et al., 2019). In the migration assay, the movement of cells across a vacant area (like a scratch or wound) is measured. A second method to measure “cell migration” is to quantify the movement of cancer cells across a transwell filter (pore size of 8 µm) from the apical chamber to the basolateral chamber. Taken together, the migration assay measures the chemotactic capability of cells to travel toward a chemo-attractant or mixture of chemo-attractants (as observed with the use of conditioned medium). The invasion assay measures both cell chemotaxis and the invasion of cells through the layer of extracellular matrix proteins, a process that is commonly found in cancer metastasis. In the “invasion assay”, the transwell filters are coated with a thin layer of basement membrane (usually Matrigel). The first event in the “invasion assay” is that the cancer cells in the apical chamber secrete matrix metalloproteases to degrade basement membrane proteins, producing a gap in the basement membrane. Subsequently, the cancer cells travel through this gap in the basement membrane and pass through the transwell filter (pore size of 8 µm), to arrive at the basolateral chamber of the transwell filter. The process through which cancer cells degrade the basement membrane to penetrate the transwell insert is a unique feature of the “invasion assay”, which is not present in “migration assays”. The invasion assay faithfully recapitulates the process of “tumor invasion” during metastasis, where the neoplastic cells degrade the basement membrane to launch themselves into circulation in the neighboring blood vessel/lymph node. It would be useful to have a second invasion assay that could validate the results obtained with the Boyden chamber assay. All these considerations led us to develop a novel method of measuring tumor invasion namely the spherical invasion assay (SIA) in our laboratory. The SIA measures the invasion of human cancer cells as they migrate from the primary Matrigel layer, over the interface, and travel into a secondary Matrigel layer. The SIA mirrors the actual process of invasion under physiological conditions (Hurley et al., 2017). The cells that grow in extracellular matrix (ECM) retain biological characteristics of tumors, such as responsiveness to diffusion gradient of oxygen, nutrients, and pH. The growth of the cells inside the ECM allows for complex cell-cell and cell-matrix interaction. The SIA can be adapted to organoids, spheroids (Gunti et al., 2021), retinal angiogenic sprouts (Stitt et al., 2005), tumor stem cells (Atashzar et al., 2020), neurospheres (da Silva Siqueira et al., 2021), and cells grown on polymeric scaffolds (Stratton et al., 2016). In such cases, it is important to remember that the conditions for culturing normal cells are very different from cancer cells. In addition, normal cells should be used at a low passage number, so that they do not become senescent. For example, if the SIA is performed with retinal endothelial cells, then the assay should be performed between passages 3-6 (Stitt et al., 2005). Furthermore, these invading cells can be characterized by confocal laser microscopy and transmission electron microscopy. Most importantly, the results of the SIA correlate with the data obtained in the Boyden chamber assay, so the SIA can be used to confirm the results obtained in Boyden chamber assays (Hurley et al., 2017). The present protocol examines the effect of the Src Kinase inhibitor PP2 (Hanke et al., 1996) (at a concentration of 10 µM) on the invasion of A549 human NSCLC cells. We hope that the SIA will be a useful tool for researchers working in the field of tumor microenvironment biology and cancer metastasis.