Gene expression in tumor cells and stroma in dsRed 4T1 tumors in eGFP-expressing mice with and without enhanced oxygenation

Cell line and culture conditions

The murine mammary cell line 4T1 (American Type Culture Collection, Rockville, MD, USA) was transfected with red fluorescent protein using a dsRed-expressing lentiviral vector. This cell line was originally isolated from a spontaneously arising mammary tumor in BALB/cfC3H mice [18]. Successful transfection with dsRed was confirmed by fluorescence microscopy (Axiolmager 2, Carl Zeiss MicroImaging, GmbH, Jena, Germany). 4T1 cells were cultured in RPMI-1640 medium (Bio-Whittaker, Verviers, Belgium) supplemented with 10% Foetal Calf Serum (Sigma-Aldrich, Steinheim, Germany), 100 units/ml penicillin, 100 μg/ml streptomycin, 2% L-glutamine (All from Bio-Whittaker, Verviers, Belgium). The cells were maintained at 37°C in 5% CO₂ and 95% air, and were seeded and used at ~ 80% confluence in all experiments.

Ethics Statement

All the animal experiments were performed in accordance with the regulations of the Norwegian State Committee for Animal Research ("Forsøkdyrutvalget", approval number 1279) and approved by the Local Institution Board at the University of Bergen (approval number 2008076BB). The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

In vivo experiments

Female NOD/SCID mice (18-24 g) were used in this study. Generally, the NOD/SCID mice used expressed eGFP in all nucleated cells, the exception being histological and immunohistochemical experiments, where plain NOD/SCID mice were used. The emission of green fluorescence has previously been observed in muscle, pancreas, kidney, heart and other organs of the mice, confirming the fluorescent phenotype [6]. The breeding was performed at the animal facility at the University of Bergen, as described by Niclou et al. [6]. All animal experiments were performed under Isoflurane (Rhone-Puolenc Chemicals, France) and N₂O gas-anaesthesia. A 17β-estradiol pellet (0.18 mg/pellet--60 day release, Innovative Research of America, Sarasota, FL, USA) was inserted into the interscapular area of all mice prior to tumor cell injection. The pellets provide a continuous release of estradiol to give serum concentrations of 150-250 pM. 3 × 10⁶ 4T1 cells dissolved in 0.15 ml PBS were injected in the mouse mammary fat pad, in the groin area. 4T1 tumors had a 100% take rate, and a 6 days latency period to a palpable tumor estimated to be ~5 mm in diameter.

The tumors were measured externally with a calliper at day 1 (pre HBO exposure), day 4 and 8 (post HBO exposure). The location of the tumor excluded external measurement in more than two dimensions. The tumor volume was therefore calculated assuming a cylindrical shape of the tumor, according to the formula: π/6 ·a²·b, where a is the shortest and b is the longest transversal diameter.

HBO treatment

A 27 l Hyperbaric Animal Research Chamber (OXYCOM 250 ARC, HYPCOMOY, Tampere, Finland) with an inner diameter of 25 cm, and an inner length of 55 cm was used. The chamber was supplied with pure O₂. After reaching 100% O₂ (15 min), the pressure was raised over a period of approximately 5 min to 2.5 bar (equivalent to 15 meters sea water). The 2.5 bar pure oxygen atmosphere was maintained for a period of 90 min. To maintain > 97% O₂ atmosphere, the chamber was flushed with pure oxygen for 3-5 min every 10-30 min depending on the number of mice in the chamber. After treatment, the mice were slowly decompressed over a 10 min period.

Three separate groups of mice were studied. The first group of tumor bearing mice was exposed to intermittent HBO treatment (2.5 bar and 100% O₂, 3 exposures à 90 min on day 1, 4 and 7). The second group was exposed to daily HBO treatments (2.5 bar and 100% O₂, 7 daily exposures à 90 min), whereas the control group was housed under normal atmosphere for the experimental period of 8 days.

In situ and ex vivo imaging

We used a fluorescence dissection microscope (Model C-DSD230, Nikon, Japan) with UV-filter optics for dsRed and eGFP, to observe the tumor in situ. After the mice were sacrificed with saturated KCl during anaesthesia, the tumors were excised. The tumors were processed in three different ways: 1) formalin (4%), and later embedded in paraffin. 2) frozen in liquid nitrogen and stored at -80°C until further use. 3) paraformaldehyde (PFA) prior to freezing, and then embedded in Prolong Gold (Invitrogen, CA, USA) after sectioning. PFA fix was performed to conserve the fluorescent traits, when visualized under the microscope (Leica TCS SP5, Wetzlar, Germany).

Histology and immunohistochemistry

Paraffin embedded tumor sections from all three groups were H&E stained, and examined by an experienced pathologist.

Frozen tumor sections (20 μm) were used for immunostaining of blood vessels using rat anti-mouse CD31 (AbD Serotec, Morphosys UK Ltd, Oxford, UK) as primary antibody and biotinylated rabbit anti-rat as secondary antibody (Vectastatin ABC kit, peroxidase IgG PK 4004, Bioteam AS, Trondheim, Norway). An ABC kit (Vectastatin ABC kit, peroxidase IgG PK 4004, Bioteam AS, Trondheim, Norway) and Diaminobenzidine tetrahydrochloride (3.3 DAB, Sigma Aldrich, Germany) were used as a chromogen to visualize blood vessels. Richardson stain was used as a nuclear counterstain. The cross-sectional density of CD31 positive structures was quantified per mm² using a counter grid, covering the viable tumor area. Tumor cell proliferation was assessed by staining with an anti-rabbit Ki67 antibody diluted 1:100 (Millipore, Billerica, MA), and biotinylated goat anti-rabbit secondary antibody (DACO Patts, Glostrup, Denmark) on frozen tumor sections. Four Ki67 labelled "hot spots" within selected high power fields of view (HPFs) were quantified using the NIS-Elements^™ BR 3.1 software (Nikon Corporation, Tokyo, Japan), under 400× magnification. The immunopositive cells were counted and expressed as a fraction (%) of the total cells. Four fields of vision per section from each animal were included in the quantification. Cell death was examined by the terminal transferase-mediated dUTP nick-end-labeling (TUNEL) method (Boehringer Mannheim, Mannheim, Germany), performed according to the manufacturers recommendation on frozen tumor sections. For quantifying TUNEL labelled cells, threshold levels of pixel intensity were determined and expressed as % positive cells/area fraction at x200 magnification. All sections were examined using a Nikon light microscope (THP Eclipse E600, Nikon Corporation) and the images were captured with a Nikon Digital Camera (DXM 1,200F, Nikon Corporation). The quantification was performed blindly.

Fluorescence-activated cell sorting (FACS)

Freshly isolated 4T1 tumors were dissociated by mincing the tissue with scalpels, followed by incubation with 1 mg/ml collagenase/dispatase (Roche Diagnostics GmbH, Germany) and 0.125% DNase I (Sigma-Aldrich) dissolved in DMEM medium for 60-90 min at 37°C. Incompletely dissociated tissue was digested a second time using the same procedure. The dissociated tumor tissue was then washed with ice-cold FACS buffer (PBS with 2% FBS) and filtered twice through a 70-μm cell strainer. The cell suspension was then centrifuged at a speed of 500 G for 10 min (4°C). The cell pellets were resuspended in FACS buffer for further analysis and sorting. The single cell suspension was filtered through a 40-μm cell strainer in order to remove any clumping cells before sorting. The cells were sorted using a cell sorter (FACS Aria SORP, BD Biosciences, Erembodegem, Belgium) based on the single-cell viability and the fluorescence intensity of eGFP and dsRed, and separation was confirmed by fluorescence microscopy (Nikon ellipse 2000, Nikon, Japan).

RNA isolation and quantification

Sorted cells were lysed using 350 μl Buffer RLT Plus with β-mercaptoethanol (β-ME). The lysate was homogenized by passing it 5 times through a 23 G needle. Further, the protocol for purification of total RNA from animal cells (RNeasy Plus Mini Handbook, Qiagen AB, Sweden) was used according to the manufacturer's recommendations. RNA concentration (ng/μl) and purity (260/280 ratio) were determined using NanoDrop 1,000 Spectrophotometer (Thermo Scientific, Sweden).

Gene expression analysis

Global gene expression analysis was performed in order to identify key molecular mechanisms and differences in gene programs in stromal and tumor cells, as well as changes following HBO treatment. A total of 18 tumor bearing mice were used. Seven mice served as control animals (non-HBO exposure), 5 mice were HBO treated intermittently and 6 mice treated daily. Stromal cells and tumor cells from the different tumors were isolated as described above. Total RNA purification, cRNA labeling, microarray hybridization and features extraction were performed as previously described [14]. The Agilent G4122F Whole Mouse Genome (4 × 44 k) Oligo Microarray Kit with SurePrint Technology (Agilent Technologies, Inc., Palo Alto, CA) was used to analyze samples in the present study. The normalized channel values were log (2) transformed and combined into a gene expression data matrix. Data were formatted in a J-Express-file suitable for additional data mining (http://www.molmine.com/) [19]. Following normalization, the treated tumor cells and stromal cells were analysed against the respective non-treated controls of stromal and tumor cells. We also analysed untreated stromal cells against untreated tumor cells to highlight and characterize the stromal cell signatures. A similar approach was performed to analyse how the treated tumor cells responded to HBO treatment versus treated stromal cells (results not shown).

Statistical analysis

For the gene expression data, we used analysis of variance (ANOVA), SAM (Significant Analysis of Microarray), GSEA (Gene Sets Enrichment Analysis) [20] and Gaussian kernels of the J-Express program package for identification of differentially expressed genes. Following SAM we selected genes with False Discovery Rate (FDR) < 5% as basis for GSEA and Hierarchical clustering. Gene sets consisting of more than 18 genes and less than 200 genes were selected for further analysis of cellular processes, pathways and molecular function. Annotated microarray data were uploaded in the BASE database and formatted and exported to ArrayExpress at the European Bioinformatics Institute (http://www.ebi.ac.uk/arrayexpress (Accession number: E-TABM-1103)) in agreement with the MIAME guidelines. Two-tailed unpaired t-test (normalized data) or the non-parametric Mann-Whitney test (non-normalized data) was used for testing the statistical differences between groups. P < 0.05 was considered statistically significant. GraphPad InStat 3 (GraphPad Software, Inc., La Jolla, USA) and SPSS for Windows (IBM Corporation, NY, USA) were used for the statistical analysis.

Fluorescent imaging

To verify tumor growth in the eGFP mice (Figure 1A), a fluorescence dissection microscope with UV-filter optics for dsRed and eGFP was used. As shown in Figure 1B, a red 4T1 mammary tumor was seen within the surrounding green host tissue. The overall tumor surface architecture was visualized, and blood vessels within the tumor bed appeared darker. The vasculature in the skin flap as well as the transition areas between tumor tissue and host tissue was also clearly visualized. Thus, dsRed transfected 4T1 mammary tumors were successfully established subcutaneously (sc) in the eGFP mice. Confocal microscopy revealed that the tumors contained both red tumor cells as well as infiltrating eGFP expressing stromal cells (Figure 1C and 1D). Before separating the tumor cells from the stromal cells using FACS, the tumor bulk was dissociated (Figure 1E). Further, a successful separation of the green stromal cells from the red tumor cells was confirmed by fluorescence microscopy (Figure 1F).

Tumor growth

Both untreated and HBO treated 4T1 mammary tumors were measured. Six days after injection of the 4T1 cells, the average size of all tumors were approximately 80 mm³ (day 1). The control tumors (n = 19) increased in size with ~800% within the first 8 days of tumor development (Figure 2). Exposing the mice to 2.5 bar pure oxygen (n = 24), on days 1, 4 and 7, each for 90 min, inhibited tumor growth significantly compared to untreated control tumors during the same time period (p < 0.001) (Figure 2). A similar reduction was found after daily HBO exposure (n = 9) 7 times (p < 0.001). Thus, enhanced oxygenation significantly inhibited the growth of the 4T1 mammary tumors.

Tumor morphology

Control 4T1 tumors (n = 7) were highly cellular and revealed an undifferentiated morphology showing both epitheloid and spindle shaped tumor cells, marked nuclear atypia and numerous mitotic figures, with diffuse growth in the surrounding fat tissue. They also showed minimal to extensive (~ 70-80%) necrosis with areas of granulation tissue. In the tumors treated with intermittent HBO (n = 5) and daily HBO (n = 9), no clear differences from control tumors were evident.

Tumor blood vessels

Cell death and proliferation

The balance between cell proliferation and cell death is an important factor deciding the tumor growth rate. We therefore aimed to investigate the amount of cell death by using TUNEL staining and proliferation by using Ki67 staining. The results showed that there were no significant differences between the three experimental groups, neither in proliferation nor cell death, when counted in hot spot areas (Table 1).

Gene expression analysis

The purity of sorted cells was verified by fluorescence microscopy. Additionally, gene expression profiling demonstrated clear separation based on the expression of mesenchymal markers in the stromal cells (Table 2). Additional file 1: Figure S1 displays the number of genes significantly changed after treatment compared to controls. 5,195 genes in the tumor cell compartment were changed both following HBO daily and intermittent treatment. In the stromal cell compartment, 4,802 genes were changed following both treatments. Additional file 2: Table S1, Additional file 3: Table S2, Additional file 4: Table S3, Additional file 5: Table S4 summarize the results from a Gene Set Enrichment Analysis (GSEA), clustering the important signalling pathways significantly changed in tumor cells and stromal cells after intermittent and daily HBO treatment, respectively. The expression of angiogenic markers in both tumor and stromal cells after intermittent and daily HBO were compared to untreated tumor and stromal cells (Table 3), demonstrating that in this study, enhanced oxygenation induced a down-regulation of pro-angiogenic genes both in tumor and stromal cells in the intermittent group. Moreover, in the daily HBO treated group, expression of pro-angiogenic genes was down-regulated in tumor cells, although fewer genes were significantly changed. Figure 3 displays a heat map of the VEGF-signalling pathway (HSA04370), showing that this important pro-angiogenic pathway is down-regulated in tumor cells after intermittent HBO, but not significantly changed in tumor cells after daily HBO. The MAPK pathway (HSA04010) was significantly down-regulated in both tumor and stromal cells after daily and intermittent HBO compared to untreated control (Figure 4a and 4b).

Epithelial markers in stroma cells		Mesenchymal markers in stromal cells
Krt14	0.3	Snai1	2
Cdh1	0.2	Snai2	19
Perp	0.3	Fgfr1	7
Dsp	0.1	Cdh2	2.3
Ocln	0.2	Cdh11	16
Cdh3	1.3	Twist2	25
		Nid1	115

The numerals are referred to as fold change in gene expression of untreated stromal cells as compared to untreated tumor cells. All values: FDR < 0.00%

		Intermittent HBO		Daily HBO
		Tumor	Stroma	Tumor	Stroma
Genes	Gene name	Fold change	Fold change	Fold change	Fold change
Anti-Angiogenic
Serpinb5	Serine (or cysteine) peptidase inhibitor, clade B, member 5	3.4	1.6	5.0	NS
Csta	Cystatin A	0.1	NS	NS	NS
Thbs1	Thrombospondin1	0.5	0.5	NS	NS
Thbs2	Thrombospondin2	NS	2.7	NS	2.5
Thbs3	Thrombospondin3	NS	NS	0.7	NS
Thbs4	Thrombospondin4	0.8	NS	NS	NS
Pro-angiogenic
HIF1a	Hypoxia-inducible factor 1, alpha	NS	NS	0.7	NS
Pecam1	Platlet/endothelial cell adhesion molecule 1	0.9	0.5	0.6	NS
Vwf	Von Willebrand factor homolog	0.7	0.3	NS	0.6
Vegfa	Vascular endothelial growth factor A	0.5	0.5	NS	1.4
Vegfb	Vascular endothelial growth factor B	NS	NS	0.7	NS
Vegfc	Vascular endothelial growth factor C	0.6	NS	0.5	NS
Pdgfa	Platelet derived growth factor, alpha	0.8	0.7	NS	NS
Pdgfb	Platelet derived growth factor, b	1.2	0.7	NS	NS
Pdgfra	Platelet derived growth factor receptor, alpha	0.7	2.3	0.5	2.1
Tgfb1	Transforming growth factor, beta 1	NS	0.8	0.8	NS
Il6	Interleukin 6	0.5	NS	NS	0.3
Fgfr1	Fibroblast growth factor receptor 1	0.9	NS	NS	NS
Fgfr3	Fibroblast growth factor receptor 3	0.7	NS	0.7	NS
Egfr	Epidermal growth factor receptor	0.5	NS	0.5	2.7
Serpinb2	Serine (or cysteine) peptidase inhibitor, clade B, member 2	0.6	NS	NS	0.5

Displayed results are fold change of gene expression in intermittent and daily hyperbaric oxygen (HBO) treated tumors cells compared to untreated control tumor cells, and intermittent and daily HBO treated stromal cells compared to untreated stromal cells. A positive fold change indicates increased expression after treatment. FDR < 0.05%, NS = not significant

Tumor and stroma in untreated control tumors

Proliferation, survival and migration of breast cancer cells can be modulated by stromal cells [22]. Microarray studies have made it possible to molecularly classify breast cancers, and correlate their signatures with metastatic behaviour and clinical outcome [23]. Nevertheless, since most of the in vivo studies have been performed on tumor tissue homogenates that provides material based on all cell types it does not allow for separate assessment of the tumor cells and stromal compartments in solid tumors, and so, subtle changes within one compartment may be masked by the bulk of surrounding cells. Thus, by using red 4T1 cells injected in green mice, we were able to investigate complex tumor-stroma interactions, making a novel contribution to the studies of tumor-host interactions in mammary tumors.

We have developed a mammary tumor model in immunodeficient mice in order to separate the stromal cells from the tumor cells, as indicated in Figure 1B. Fluorescence microscopy (Figure 1F) showed a successful separation of the green stromal cells from the red tumor cells by FACS. Stromal cells expressed mesenchymal markers, as indicated in Table 2, confirming their origin. Gene expression results in the untreated control tumors also verify expression-patterns of stromal cells, with high expression of ECM genes, such as collagens and integrins, in addition to high expression of MMP's and chemokines involved in invasion and metastasis (data not shown).

Changes in tumor and stroma after enhanced oxygenation

The present study aimed to elucidate the effect of enhanced oxygenation on tumor growth and tumor-stroma interactions. Previous studies from our laboratory have shown a significant tumor inhibitory effect of intermittent HBO on DMBA (dimetyl-α-benzantracene)-induced mammary tumors [12, 14, 16]. Thus, we wanted to expand our study of enhanced oxygenation to another mammary tumor model, to determine if HBO has a general inhibitory effect on breast cancer. Both treatment regimens had a significant growth-inhibitory effect on the present 4T1 tumor model. However, the inhibitory effect was not as pronounced in the present 4T1 tumors as for the DMBA-induced tumors [12, 14, 16].

The number of genes significantly changed after treatment compared to control is shown in Additional file 1: Figure S1. To be able to systematize the gene expression results, we have searched for important signalling pathways by GSEA. Signalling pathways significantly changed in tumor cells and stromal cells after intermittent and daily HBO treatment, respectively, is shown in Additional file 2: Table S1, Additional file 3: Table S2, Additional file 4: Table S3, Additional file 5: Table S4. The results will be discussed further in the following sections.