Steele Lab

It has been shown in our lab that solid stress from the surrounding matrix inhibits the growth of tumor spheroids in vitro [1] and that the stress may induce the expression of hyaluronan which facilitates cell-cell adhesion [2]. It was assumed that the plateau phase in spheroid size observed in those studies was due to the counterforce from the increasingly compressed agarose gels. Yet it is not clear if that is true and, if it is, how the solid stress transduces in the agarose gels. Another related question is how the stress distributes inside the spheroids. The goal of this project is to find quantitative answers to these questions.

Methods

For the measurement of solid stress transduction in agarose gels, 1 mm red fluorescent latex beads are co-embedded with GFP-transfected human colon adenocarcinoma cells (LS174T) in agarose gels of different densities (0.3%, 0.5%, 0.75% and 1.0%). Tumor spheroids are tracked using gridded cover slips that are fixed to the bottom of dishes containing the spheroids and gels. Confocal images of spheroids and their surrounding micro-beads (Figure 1) are taken every 7 days for 35 days. The images are analyzed using a Matlab program that calculates the average density of beads as a function of distance from the spheroid surface.

Figure 1

For the measurement of solid stress distribution inside the spheroids, cell density is used as a quantitative indicator of the stress level. At different time points, spheroids (different from those tracked during the measurement of solid stress distribution in agarose gels) are first stained with Hoechst 33324, a blue fluorescent nuclei dye. Stacks of GFP and DAPI confocal images of 20 spheroids are then taken for each gel density. The GFP images (Figure 2A) give the volume of the spheroid while the DAPI images (Figure 2B) reveal the number of cells it harbors. From those data, we can get an estimate of cell density as the function of location inside the spheroid, gel density and time.

Figure 2A

Figure 2B

Results

Typical distribution of beads around spheroids at Day 7 in 0.5% (Figure 3A) and 1.0% (Figure 3B) agarose gels:

Figure 3a

Figure 3b

References

[1] Helmlinger G, Netti PA, Lichtenbeld HC, Melder RJ, Jain RK. Solid stress inhibits the growth of multicellular tumor spheroids. Nat Biotechnol. 1997 Aug;15(8):778-83.

[2] Koike C, McKee TD, Pluen A, Ramanujan S, Burton K, Munn LL, Boucher Y, Jain RK. Solid stress facilitates spheroid formation: potential involvement of hyaluronan. Br J Cancer. 2002 Mar 18;86(6):947-53.