Stiffened Extracellular Matrix and Signaling from Stromal Fibroblasts via Osteoprotegerin Regulate Tumor Cell Invasion in a 3-D Tumor in Situ Model

Abstract

Several changes have been described in the stroma surrounding a tumor, including changes in cellular composition, altered extracellular matrix composition and organization, and increases in stiffness. Tumor cells are influenced by the composition, organization, and mechanical properties of the microenvironment, and by signals from stromal cells. Here we sought to test whether signaling from stromal fibroblasts and/or the small change in stiffness observed in vivo surrounding epithelial tumors regulates tumor cell invasion from a model of a tumor in situ. We generated a novel tumor in situ model system in which a tumor spheroid is encased within a collagen-IV containing membrane and further encased within a collagen-I matrix of in vivo stiffness with or without fibroblasts. Effects of the matrix, fibroblasts or fibroblast signals were determined by observing the invasion of tumor cells into the matrix. Effects of reciprocal tumor cell signaling upon fibroblasts were determined by observing markers of fibroblast activation. We found that a stiffened matrix led to increased dissemination of MDA-MB-231 cells from tumor spheroids when no fibroblasts were present and that MCF10A cells maintained a more normal organization with a stiffened matrix. The presence of fibroblasts, or fibroblast conditioned media, attenuated the effect upon MDA-MB-231 cells. We also observed an attenuation of fibroblast activation associated gene expression in the presence of MDA-MB-231 cells, with a paradoxical increase in activation associated contractile activity. Furthermore, we identified osteoprotegerin as a soluble factor released by fibroblasts in the stiffened environment that is key to the inhibition of cell invasion.

Keywords: 3D hydrogel; Extracellular matrix; Invasion; Mechanotransduction; Tumor in situ.

Fig. 1

Spheroid characterization. a Spheroid size. Asterisk indicates significant difference. b Spheroid circularity as measured by Fiji (ImageJ) software [33]. Circularity is a measure of the fit of the measured shape to that of a circle, calculated as 4π(area/perimeter²). A value of 1.0 indicates a perfect circle whereas approaching 0 indicates an elongated polygon. Asterisk indicates significant difference. ANOVA p-values <0.05. Error bars show standard error of the mean. c & d Representative confocal images of MDA-MB-231 (c) and MCF10A (d) spheroids directly after incorporation into a collagen-I hydrogel and immunostained for collagen-IV. Right panels represent 5 μm optical sections in the Z dimension. Scale Bar =100 μm

Fig. 2

Dissemination and size of spheroids. a Number of MCF10A or MDA-MB-231 cells that have dissociated from the main tumor spheroid after 3 days of culture. Disseminated cells were counted as cells which were not in contact with the main spheroid body – including disseminated clusters. b Final size of the tumor spheroid after 3 days of culture. c Representative phase contrast images of spheroids at day 0 and day 3. Images are of the same spheroid at day 0 and day 3. d Day 3 staining of collagen-IV is reduced in stiff hydrogels. e Quantification of collagen-IV staining. Asterisk indicates significant difference from day 0; Student’s t-test p-value <0.05. Error bars show standard error of the mean. All scale bars are 100 μm

Fig. 3

Dissemination and spheroid size of MDA-MB-231 spheroids cultured with fibroblasts. a Hydrogel compaction over 3 days. Asterisk indicates significant increase in final size from 100 K; Hash indicates significant decrease in final size from 100 K; Student’s t-test p-value <0.05. b Semi-quantitative immunocytochemistry of αSMA. c Semi-quantitative immunocytochemistry of palladin. Values normalized to soft collagen-I hydrogel with spheroid present. Asterisk indicates significant difference from spheroid in soft gel. Double asterisk indicates significant difference from spheroid in soft gel and no spheroid in stiff gel; Student’s t-test p-value <0.05. d Number of cells which have dissociated from the main tumor spheroid. Asterisk indicates significant difference from day 0; Student’s t-test p-value <0.05. Double asterisk indicates significant difference from day 0; Student’s t-test p-value <0.005. (E) Final size of the tumor spheroid. Asterisk indicates significant difference from day 3 control; Student’s t-test p-value <0.05. Error bars signify standard error of the mean

Fig. 4

Fibroblast secreted cytokines override stiffness response. a Number of cells which have dissociated from the main tumor spheroid. For reference, diamond in plot is spheroid disassociation with 50 K fibroblasts present in hydrogel (Fig. 3). Asterisk indicates significant difference from day 0; Student’s t-test p-value <0.05. Double asterisk indicates significant difference from day 0; Student’s t-test p-value <0.005. b Final size of the tumor spheroid. There were no statistically significant differences on day 3; Student’s t-test p-value >0.05. c Representative phase contrast images of spheroids on day 3 with conditioned media treatment. Scale Bar =200 μm

Fig. 5

Osteoprotegerin secretion is increased in activated fibroblasts and inhibits dissemination. a Quantification of cytokine array targets which were not present in or increased concentration from unconditioned media. Data normalized to unconditioned media by normalizing to array control and subtracting unconditioned media baselines. Asterisk indicates significant difference from soft gel. Student’s t-test p-value <0.05. Error bars show standard deviation. b Dissemination of cells from spheroids treated with soft hydrogel fibroblast media and additional recombinant OPG. Fewer cells disseminated from stiff stromal spheroids when OPG level was increased. Diamond markers represent spheroid dissemination when cultured within a stiff matrix with stiff matrix HFb conditioned media. c Dissemination of cells from spheroids treated with stiff hydrogel fibroblast media and an antibody to OPG. Cells disseminated from spheroids, regardless of hydrogel stiffness, when blocking OPG. Diamond markers represent spheroid dissemination when cultured within a stiff matrix with soft matrix HFb conditioned media. Asterisk indicates significant difference from conditioned media only. Student’s t-test p-value <0.05. Error bars show standard deviation of the mean