Inhibition of tumor cell proliferation and motility by fibroblasts is both contact and soluble factor dependent

Abstract

Normal human and murine fibroblasts can inhibit proliferation of tumor cells when cocultured in vitro. The inhibitory capacity varies depending on the donor and the site of origin of the fibroblast. We showed previously that effective inhibition requires formation of a morphologically intact fibroblast monolayer before seeding of the tumor cells. Here we show that inhibition is extended to motility of tumor cells and we dissect the factors responsible for these inhibitory functions. We find that inhibition is due to two different sets of molecules: (i) the extracellular matrix (ECM) and other surface proteins of the fibroblasts, which are responsible for contact-dependent inhibition of tumor cell proliferation; and (ii) soluble factors secreted by fibroblasts when confronted with tumor cells (confronted conditioned media, CCM) contribute to inhibition of tumor cell proliferation and motility. However, conditioned media (CM) obtained from fibroblasts alone (nonconfronted conditioned media, NCM) did not inhibit tumor cell proliferation and motility. In addition, quantitative PCR (Q-PCR) data show up-regulation of proinflammatory genes. Moreover, comparison of CCM and NCM with an antibody array for 507 different soluble human proteins revealed differential expression of growth differentiation factor 15, dickkopf-related protein 1, endothelial-monocyte-activating polypeptide II, ectodysplasin A2, Galectin-3, chemokine (C-X-C motif) ligand 2, Nidogen1, urokinase, and matrix metalloproteinase 3.

Keywords: cancer-associated fibroblast; extracellular matrix; motility; soluble factors; tumor microenvironment.

Fig. 1.

Conditioned medium from confronted fibroblast cultures significantly inhibited the proliferation of tumor cells. Two thousand BJhTERT whirly cells were seeded and cultured for 5–7 d to form a confluent monolayer in a 384-well plate-based inhibition assay. On day 0 of coculture, 200 PC3 mRFP tumor cells were seeded on top of the old confluent fibroblast monolayers. The inhibition of PC3 mRFP proliferation has been tested on both fixed and live monolayers of BJhTERT whirly cells and was compared with the proliferation rate when cultured alone (blue line). CCM collected from BJhTERT whirly + PC3 mRFP coculture (red line) and NCM collected from BJhTERT whirly cells cultured alone (green line) were also tested in 384-well plate-based inhibition assay. Data are presented as arcsin(sqrt(value), which represent the ratio of PC3 mRFP cells on day 5 to day 0. Factorial ANOVA followed by Tukey's HSD test were performed to determine the significance effect between group means and for each treatment.

Fig. 2.

Conditioned medium from confronted fibroblast cultures significantly inhibited the motility of tumor cells. Tumor cell motility was studied by extended field live cell microscopy. Trajectories of tumor cells growing on top of fixed BJhTERT whirly fibroblast monolayer. Trajectories of PC3 mRFP tumor cells during 12-h intervals (A) treated with confronted CM or (C) with nonconfronted CM. Color-coded images show a 60 time point projection of the red-labeled tumor cells: yellow (0–12 h), green (12–24 h), magenta (24–36 h), blue (36–48 h), and red (48–60 h). (D and B) A maximum projection of all five color-coded images shows the total motility (full trajectories) of the PC3 mRFP tumor cells during 0–60 h for BJhTERT whirly nonconfronted and confronted CM, respectively.

Fig. 3.

Kinetics of tumor cell motility and proliferation. (A) Motility of PC3 mRFP cells when treated with NCM (blue line) and CCM (red line). Motility of tumor cells has been quantified by calculating the area of cell trajectories normalized for average number of cells in 12-h intervals. (B) Number of PC3 mRFP cells proliferated during 60 h (time points). Factorial ANOVA followed by Tukey's HSD test were performed to determine the significance effect between group means for all intervals.

Fig. 4.

Conditioned medium (CM) has no effect on tumor cell motility when cultured alone without fibroblasts underneath. Tumor cell motility was studied by extended field live-cell microscopy. Trajectories of tumor cells cultured alone. Trajectories of PC3 mRFP tumor cells during 12-h intervals (C) treated with confronted CM and (A) with medium-only color-coded images show 50 time points projection of the red labeled PC3 tumor cells: yellow (0–12 h), green (12–24 h), magenta (24–36 h), blue (36–48 h) and red (48–60 h). (D and B) A maximum projection of all five color-coded images shows the total motility (full trajectories) of the PC3m RFP tumor cells during 0–60 h for confronted CM and medium only, respectively.

Fig. 5.

Different soluble proteins were identified in CM of fibroblast cultures. The 507 different soluble factors were analyzed using L-Series Human Antibody Array L-507 Membrane Kit (RayBiotech) in nonconfronted BJhTERT CM (A), confronted BJhTERT + PC3 CM (B), and PC3 alone CM (C). Proteins that showed up-regulation after confrontation are highlighted in rectangular frame. Protein that showed down-regulation after confrontation is highlighted in an oval frame. All membranes have been visualized in 30-s exposure time. GDF-15 (1), DKK1 (2), EDA-A2 (3), EMAP-II (4), Galectin-3 (5), CXCL2 (6), Nidogen1 (7), uPA (8), and MMP3 (9).

Fig. 6.

Running hypothesis for fibroblast inhibition of tumor cell proliferation. The inhibition of tumor cell proliferation and motility requires both structure molecules (surface receptors and ECM) as first signal and the soluble factors secreted after fibroblast-tumor cells cross-talk as a second signal.