Solid stress facilitates spheroid formation: potential involvement of hyaluronan

Abstract

When neoplastic cells grow in confined spaces in vivo, they exert a finite force on the surrounding tissue resulting in the generation of solid stress. By growing multicellular spheroids in agarose gels of defined mechanical properties, we have recently shown that solid stress inhibits the growth of spheroids and that this growth-inhibiting stress ranges from 45 to 120 mmHg. Here we show that solid stress facilitates the formation of spheroids in the highly metastatic Dunning R3327 rat prostate carcinoma AT3.1 cells, which predominantly do not grow as spheroids in free suspension. The maximum size and the growth rate of the resulting spheroids decreased with increasing stress. Relieving solid stress by enzymatic digestion of gels resulted in gradual loss of spheroidal morphology in 8 days. In contrast, the low metastatic variant AT2.1 cells, which grow as spheroids in free suspension as well as in the gels, maintained their spheroidal morphology even after stress removal. Histological examination revealed that most cells in AT2.1 spheroids are in close apposition whereas a regular matrix separates the cells in the AT3.1 gel spheroids. Staining with the hyaluronan binding protein revealed that the matrix between AT3.1 cells in agarose contained hyaluronan, while AT3.1 cells had negligible or no hyaluronan when grown in free suspension. Hyaluronan was found to be present in both free suspensions and agarose gel spheroids of AT2.1. We suggest that cell-cell adhesion may be adequate for spheroid formation, whereas solid stress may be required to form spheroids when cell-matrix adhesion is predominant. These findings have significant implications for tumour growth, invasion and metastasis.

Figure 1

AT2.1 (A) and AT3.1 (B) cultures after 30 days in agarose gels. The left and right columns show AT2.1 and 3.1, respectively, in various concentrations of agarose (0% refers to cells in culture media). Note that at the highest concentration, 1.8%, cell growth is inhibited in both cell lines. Scale bar=100 μm

Figure 2

Growth curves and Gompertz fits for AT2.1 (A) and AT3.1 (B) spheroids in gels of various concentration. Data that could not be fit with the Gompertz equation are denoted with a dotted line. Agarose concentrations are indicated on the plot. The rapid rise at the last time point for the 0% sample in (B) is likely due to loose aggregation resulting from crowding of the system rather than actual spheroid formation. (C) and (D) give the Gompertz growth parameters α (specific growth rate) and D_max (asymptomatic size), respectively. (C) Solid circles represent AT2.1 and open boxes AT3.1. (D) Open triangles represent AT2.1 and solid diamonds AT3.1.

Figure 3

Enzymatic digestion of agarose gel cultures after 30 days of growth. AT2.1 and AT3.1 are shown in the left and right columns, respectively. Images shown are from 3, 6, and 8 days after release of spheroids from gel. Note that the AT3.1 cells return to a dis-aggregated state, but the AT2.1 maintain their spheroidal morphology at 8 days after release. Scale bar=100 μm.

Figure 4

Histological sections of spheroids in agarose gels. AT3.1 cells in agarose, are loosely-packed with a uniform matrix space observed between the cells (A). On the other hand, in spheroids of AT2.1 (B) and aggregates of AT3.1 (C) there is no visible matrix between most of the cells. Scale bar=20 μm.

Figure 5

Spheroid sections in gels and in free suspension stained with the hyaluronan binding protein. Spheroids of AT3.1 grown in 1% agarose gels (A), AT3.1 aggregates in free suspension (B) and AT2.1 spheroids in 1% agarose gels (C). Brown staining indicates the presence of hyaluronan, while nucleii are stained blue (haematoxylin). In AT3.1 spheroids in agarose (A), hyaluronan staining is localised between the cells and at the spheroid–agarose interface. In contrast, the hyaluronan staining is not observed in free suspension aggregates of AT3.1 (B). Hyaluronan is associated with the surface of tumour cells or appears as globular structures in AT2.1 spheroids (C). Scale bar=25 μm.