Figure 1.
Integrin α11 expression on MEFs affects heterospheroid size.
(A) Schematic illustration of spheroid formation: Spheroids were grown from fibroblast cells alone (homospheroids), or mixed with A549 tumor cells in a ratio of 4∶1 (MEFs: A549) to form heterospheroids. (B) Representative phase contrast images of heterospheroids generated by the hanging drop method after 4 days in culture. Heterospheroids composed of α11-/- MEFs formed larger spheroids. The size difference is significant between the α11-/-/A549 and the α11-containing spheroids (α11+/+/A549 and α11-/-/α11/A549). Size bars = 100 µm; ****p< 0.0001. (C) Western-blot analysis of α11 integrin expression level during spheroid culture at the indicated time points. The lower panel shows the α11 integrin expression levels after normalizing to β-actin expression levels.
Figure 2.
Absence of α11 expression on MEFs leads to impaired ability to contract collagen I lattices and decreased interstitial fluid pressure in heterospheroids.
(A) Heterospheroids of MEFs of different genotypes and A549 (as indicated) were fixed and stained with an antibody towards mouse collagen type I (red). Nuclei were counterstained with DAPI (blue). Size bars = 100 µm. (B) A549 cells or the MEFs and A549 cell mixtures (MEFs+A549, ratio 4∶1) were embedded in type I collagen as indicated. Contraction of collagen gels was monitored at 2, 4, 8 and 20 hours. Three independent experiments were performed and data shown are from one representative experiment with 6-8 replicates for each cell type or cell mixture. Error bars represent standard deviation. (C) Interstitial fluid pressure measurement of the spheroids. The interstitial fluid pressure (Pif) was measured in spheroids cultured for 4 and 6 days. The numbers of spheroids measured (n) are as indicated. **** p< 0.0001.
Figure 3.
A549 cell segregation and proliferation inside the spheroids.
(A) Four- day-old heterospheroids (as indicated) were double-stained with anti-human cytokeratin 7 (stained A549 cells, red) and anti-mouse β1 integrin (stained MEFs, green) antibodies. Size bars = 100 µm. (B) A549 proliferation in the spheroids. Spheroids were prepared with different types of MEFs and A549 cells stably expressing the firefly luciferase (A549-Luc). A549 proliferation was measured by luciferase assay on spheroids at different time points. Each experiment was performed with spheroid collected from three separate preparations at each time point and measured in duplicte. Experiments were repeated for at least three times and shown is the representative result from one experiment. Error bars indicate standard deviation. ** p<0.01, *** p<0.001.
Figure 4.
Spheroid migration on collagen I monolayers and invasion in 3D collagen gels.
(A) A549 migration out from the heterospheroids. Six-day-old heterospheroids were seeded on collagen type I-coated coverslips and cultured under conditions as described in Materials and Methods. Migration of the cells from the spheroids was examined under an inverted phase contrast microscope 4 and 24 hours after the spheroids were seeded. Size bars = 100 µm. (B) Fluorescence immunostaining of the spheroids on coverslips after 24 h and migration distance of A549 from the heterospheroids. Spheroids were double stained with antibodies towards human keratin 7 (stained A549 cells, red) and mouse β1 integrin (stained MEFs, green). Six of each type of spheroids were stained. The maximum migration distance (d) of A549 cells were measured on the photographs as indicated in the figure and calculated according to size bar. Size bars = 100 µm. **** p<0.0001. (C) Spheroid invasion in 3D collagen I gel. Six to eight 6-day-old spheroids were embedded in 3D collagen gels (1 mg/ml collagen type I) and invasion of cells was observed and photographed under an inverted phase contrast microscope after 24 h and 48 h. Two representative images are shown. Size bars = 100 µm.
Figure 5.
A549 expression profiling in α11-/-/A549 and α11+/+/A549 heterospheroids.
(A) The top 10 up- and down-regulated genes in A549 cells in α11-/-/A549 spheroids compared with those in α11+/+/A549 spheroids. Fold changes shown are the average values from 6 individual samples from each type of spheroid. Gene symbols marked in bold are the genes chosen for RT-qPCR verification. (B) Microarray data verification of two selected genes using qRT-PCR. Total RNA was extracted from 6-day-old spheroids prepared in new replicate experiments in conditions identical to those used for Microarray (n = 6). Shown is the fold change of the mRNA expression levels of the genes in α11-/-/A549 comparing with those in α11+/+/A549 spheroids (set arbitrarily as 1). Error bars represent standard deviation across 6 independent samples.
Figure 6.
CXCL5 regulates A549 proliferation in heterospheroids.
Heterospheroids were prepared with α11+/+ or α11-/- MEFs, mixed with A549 cells stably expressing firefly luciferase (A549-Luc). Proliferation of A549 cells was measured by luciferase assay in presence of CXCL5 receptor inhibitor (SB225002) in α11+/+/A549 spheroids (upper graph) and in presence of recombinant CXCL5 or SB225002 in α11-/-/A549 spheroids (lower graph). Three independent experiments were performed and results from one representative experiments are shown. Error bars represent standard deviation. * p<0.05, ** p<0.01, *** p<0.001.
Figure 7.
CXCL5 regulates heterospheroid invasion in 3D collagen gels.
Six-day-old spheroids were embedded in 3D collagen gels (1 mg/ml collagen type I) in the presence of DMSO (control) or different concentrations of the CXCL5 receptor inhibitor SB225002 (upper graph) or recombinant CXCL5 (lower graph). Six spheroids were imbedded into the collagen gels under each condition. Invasion of the cells from the spheroids into the collagen gels was observed under an inverted phase contrast microscope and photographed after 48 h. Size bar = 100 µm.