3.1 Stiffness characterization

We observed the growth of distinct necrotic centers in older spheroids, which has been reported [8, 17]. Fig 1 shows the difference in spheroid core with respect to time in day 20 (Fig 1A) and day 5 (Fig 2B,). The average sizes of T47D spheroids at day 20 and day 5, respectively, were 457±16 μm and 421±26 μm. Mechanical analysis of day 5 and day 20 spheroids was conducted for n = 6 samples using microtweezers. Fig 2 shows the compression steps of T47D day 5 (Fig 2, Panel A) and day 20 (Fig 2, Panel B) spheroids. The red squares indicate the reference positions of the cantilever tips measured without a spheroid, and the green square depicts the position of cantilever tips when the spheroid is compressed between the cantilevers. The colored squares were generated from the custom-built pattern matching software (see [29] for details). Comparing sequences A1-A3 and B1-B3 in Fig 2, one can see that the separation between the top green square and its respective red square is greater in B rather than in A. This difference shows that the cantilevers in B1-B3 were bent more than in A1-A3 because the spheroid in B1-B3 (i.e. day 20 spheroid) is stiffer than the one in A1-A3 (i.e. day5 spheroid). We found that day 20 spheroids exert ~ 2.5 times more force on average than day 5 spheroids. The average Young’s moduli of T47D day 5 and day 20 spheroid were 260±100 and 680±150 Pa, respectively, which shows day 20 spheroids are significantly stiffer (p<0.0005) than day 5 spheroids.

Example of (A) day 20 and (B) size-matched day 5 T47D spheroid. A dense center is visible in the day 20 spheroid. Day 20 spheroids were seeded from 1000 cells/ well and day 5 spheroids were seeded from 4000 cells/well to prepare size-matched samples.

Mechanical characterization of T47D day 5 (Panel A) and day 20 (Panel B) was preformed using microtweezers. The average size of spheroids was 425 μm. Day 20 spheroids exerted higher force on the microcantilevers than day 5, size matched, spheroids.