Three-Dimensional Quantification of Spheroid Degradation-Dependent Invasion and Invadopodia Formation

Abstract

Invadopodia are actin-rich, proteolytic structures that enable cancer cell to invade into the surrounding tissues. Several in vitro invasion assays have been used in the literature ranging from directional quantitative assays to complex three-dimensional (3D) analyses. One of the main limitations of these assays is the lack of quantifiable degradation-dependent invasion in a three-dimensional (3D) environment that mimics the tumor microenvironment. In this article, we describe a new invasion and degradation assay based on the currently available tumor spheroid model that allows long-term high-resolution imaging of the tumor, precise quantification, and visualization of matrix degradation and multichannel immunocytochemistry. By incorporating a degradation marker (DQ-Green BSA) into a basement-membrane matrix, we demonstrate the ability to quantitate cancer cell-induced matrix degradation in 3D. Also, we describe a technique to generate histological sections of the tumor spheroid allowing the detection of invadopodia formation in the 3D tumor spheroid. This new technique provides a clear advantage for studying cancer in vitro and will help address critical questions regarding the dynamics of cancer cell invasion.

Methods for in vitro modeling of glioma invasion: Choosing tools to meet the need

“…The sandwich invasion assay, a 3D spheroid invasion model, places a tumor neurosphere in between two layers of matrix then follows cells as they spread out from the central sphere (Figure 3b; Goertzen, Eymael, & Magalhaes, 2018;Markwell et al, 2019;Sharma, Singh, Siraj, & Saxena, 2018). Diverse ECMs can be incorporated into this assay, but most studies utilize either collagen I or Matrigel due to ease, rapid gelification, and commercial availability (Markwell et al, 2019;Naakka et al, 2019;Sharma et al, 2018).…”

“…Diverse ECMs can be incorporated into this assay, but most studies utilize either collagen I or Matrigel due to ease, rapid gelification, and commercial availability (Markwell et al, 2019;Naakka et al, 2019;Sharma et al, 2018). An acellular ECM layer is deposited into the bottom of a well then allowed to solidify (Goertzen et al, 2018;Markwell et al, 2019;Nath & Devi, 2016;Sharma et al, 2018). Next, neurospheres are suspended in the ECM mixture and placed atop the solidified ECM layer.…”

“…As the top layer solidifies, neurospheres sink down to the interface between the two ECM layers, hence the sandwich assay title as spheres are "sandwiched" between ECM layers. If the cells undergo collective invasion, this takes on a fried egg appearance where the invasive front spreads out like the egg white and the central sphere remains in the center similar to the egg yolk (Goertzen et al, 2018;Markwell et al, 2019;Sharma et al, 2018;Tevis, Colson, & Grinstaff, 2017). While theoretically spheres should spread evenly in all directions, it is not uncommon to find that cells spread out preferentially along the interface between ECM layers (Goertzen et al, 2018;Markwell et al, 2019;Sharma et al, 2018;Tevis et al, 2017).…”

“…If the cells undergo collective invasion, this takes on a fried egg appearance where the invasive front spreads out like the egg white and the central sphere remains in the center similar to the egg yolk (Goertzen et al, 2018;Markwell et al, 2019;Sharma et al, 2018;Tevis, Colson, & Grinstaff, 2017). While theoretically spheres should spread evenly in all directions, it is not uncommon to find that cells spread out preferentially along the interface between ECM layers (Goertzen et al, 2018;Markwell et al, 2019;Sharma et al, 2018;Tevis et al, 2017). Although this assay artifact may limit certain studies, there is evidence that in vivo invasion also occurs along 2D planes within the body (Holland et al, 2000;Louis, 2006;Onishi, Ichikawa, Kurozumi, & Date, 2011;Weigelin, Bakker, & Friedl, 2012 (Goertzen et al, 2018;Sand et al, 2013).…”

Investigating TNS4 in the Colorectal Tumour Microenvironment Using 3D Spheroid Models of Invasion

“…At day 3 of spheroid formation, spheroids were transfered to a chilled flat-bottomed 96-well plate coated with 50uL/well of Cultrex BME RGF (Trevigen) at 6mg/mL and overlaid with another 50uL/well of Cultrex and 100uL of complete Fluorobrite DMEM supplemented with 80ng/mL human recombinant EGF as a chemoattractant. For extracellular matrix degradation analysis, DQ BSA-Green was added to Cultrex at 30ug/mL as previously described [23] .…”