CXCL5 regulates A549 proliferation in heterospheroids.

<p>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.</p

Integrin α11 expression on MEFs affects heterospheroid size.

<p>(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.</p

A549 cell segregation and proliferation inside the spheroids.

<p>(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.</p

A549 expression profiling in α11^-/-/A549 and α11^+/+/A549 heterospheroids.

<p>(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.</p

CXCL5 regulates heterospheroid invasion in 3D collagen gels.

<p>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.</p

Spheroid migration on collagen I monolayers and invasion in 3D collagen gels.

<p>(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.</p

Phenotypes of tumor cell lines grown as single cell type tumor spheroids and fibroblast/tumor composite spheroid using the hanging drop method.

<p>Spheroids were grown from tumor cells alone (100%), or mixed with different proportions of wild type mouse embryonic fibroblasts. 50% and 10% refer to the amount of tumor cells in the composite spheroids. Spheroid morphology was assessed and photographed under a phase contrast microscope using a Nikon D3000 digital camera 4 and 6 days after seeding.</p

Proliferation.

<p>Ki67-staining revealed a significantly higher number of proliferating cells in the control group compared to the HBOT group in both models (MDA-MB-231, * p = 0.0002. BT-474, * p = 0.0001). Scale bar 500 μm.</p

Oxygen-dependent regulation of tumor growth and metastasis in human breast cancer xenografts

<div><p>Background</p><p>Tumor hypoxia is relevant for tumor growth, metabolism, resistance to chemotherapy and metastasis. We have previously shown that hyperoxia, using hyperbaric oxygen treatment (HBOT), attenuates tumor growth and shifts the phenotype from mesenchymal to epithelial (MET) in the DMBA-induced mammary tumor model. This study describes the effect of HBOT on tumor growth, angiogenesis, chemotherapy efficacy and metastasis in a triple negative MDA-MB-231 breast cancer model, and evaluates tumor growth using a triple positive BT-474 breast cancer model.</p><p>Materials and methods</p><p>5 x 10⁵ cancer cells were injected s.c. in the groin area of NOD/SCID female mice. The BT-474 group was supplied with Progesterone and Estradiol pellets 2-days prior to tumor cell injection. Mice were divided into controls (1 bar, pO₂ = 0.2 bar) or HBOT (2.5 bar, pO₂ = 2.5 bar, 90 min, every third day until termination of the experiments). Treatment effects were determined by assessment of tumor growth, proliferation (Ki67-staining), angiogenesis (CD31-staining), metastasis (immunostaining), EMT markers (western blot), stromal components collagen type I, Itgb1 and FSP1 (immunostaining) and chemotherapeutic efficacy (5FU).</p><p>Results</p><p>HBOT significantly suppressed tumor growth in both the triple positive and negative tumors, and both MDA-MB-231 and BT-474 showed a decrease in proliferation after HBOT. No differences were found in angiogenesis or 5FU efficacy between HBOT and controls. Nevertheless, HBOT significantly reduced both numbers and total area of the metastastatic lesions, as well as reduced expression of N-cadherin, Axl and collagen type I measured in the MDA-MB-231 model. No change in stromal Itgb1 and FSP1 was found in either tumor model.</p><p>Conclusion</p><p>Despite the fact that behavior and prognosis of the triple positive and negative subtypes of cancer are different, the HBOT had a similar suppressive effect on tumor growth, indicating that they share a common oxygen dependent anti-tumor mechanism. Furthermore, HBOT significantly reduced the number and area of metastatic lesions in the triple negative model as well as a significant reduction in the EMT markers N-cadherin, Axl and density of collagen type I.</p></div

Collagen type I.

<p>The total amount of collagen type I quantified as percent of total area in both control (n = 5 tumors) and HBOT (n = 5 tumors) primary tumors (A) is shown. The total amount of ITGB1 as percent of total area in both control (n = 5 tumors) and HBOT (n = 5 tumors) primary tumors (B). A representative immunofluorescence (IF) staining picture from a control (C) and HBOT (D) tumor is shown. Green represents stroma cells (stained with antibody to mouse integrin-β1) red represents collagen type I secreted by the stromal cells (stained with antibody to mouse collagen type I), and blue shows the nuclei stained with DAPI. Scale bar represents 100 μm. Representative IF pictures are also shown to illustrate the compostion/proportion of the tumor cells (red, stained with cytokeratin 7 antibody) and the stromal cells (green, stained with integrin β1 antibody) from the control (E) and and HBOT (F) tumors, respectively. Scale bar represents 100 μm.</p