Development of an inducible model of EMT using a single cell clone derived from a

Transforming Growth Factor β

3.2.1.1 Morphological changes induced in P5B3 through the treatment with 10 ng/ml TGF-β for 5 days

Transforming growth factor β, which is a known inducer of EMT in vitro, was selected for this study. The untreated cells of P5B3 present a “cobblestone” morphology of epithelial cells tightly attached to each other and the flask surface. Initially, the cells were treated with 10 ng/ml TGF-β for 5 days, which induced morphological changes compared to the untreated control (Fig. 3.4), showing a change from connected island of cells to dispersed elongated cells. The cells developed an elongated cell shape and isolation from surrounding cells. Furthermore, their adherence to the flask surface was reduced. The untreated P5B3 cells did not show any changes in their morphology nor their adherence to the cell culture flask after 5 consecutive days of growth.

Figure 3.4: Morphological changes of P5B3 after treatment with TGF-β for 5 days with 10 ng/ml TGF-β. The scale bars indicate 10 µm.

3.2.1.2 Gene expression changes induced in P5B3 through the treatment with 10 ng/ml TGF-β for 5 days

After morphological changes were observed through the treatment with TGF-β, the cells were screened for potential changes in the molecular EMT profile. For this, extracted RNA of both conditions was analysed for the following genes: VIM, CDH1, CDH2, FN1, and the EMT-Transcription factors (EMT-TFs) SNAI1, SNAI2, TWIST1 and ZEB1

86

using quantitative real-time PCR. Figure 3.5 demonstrates the expression changes of these genes at the mRNA level in treated cells compared to the natural P5B3 profile. It could be shown that the treatment induced an expression of the analysed markers associated with a mesenchymal cell state, whereas the epithelial associated gene, CDH1, showed a decreased expression. Of all the analysed genes, VIM showed the strongest increase with about 1000 times the expression compared to the untreated cells. The other mesenchymal associated genes, CDH2 and FN1, showed the second and third strongest upregulation, respectively. Additionally, the EMT-TFs all showed an increase in their expression, of which ZEB1 was showing the strongest fold change increase, induced through the treatment. Based on the detected molecular changes indicating morphological changes to an increased mesenchymal phenotype and subsequent induction of EMT, this cell line model was selected for further characterisation and analysis.

V I M C D H 1 C D H 2 F N 1 S N A I 1 S N A I 2 T W I S T 1 Z E B 1 - 5 2 0 5 0 7 5 1 0 0 5 0 0 1 0 0 0 1 5 0 0 E M T m a r k e r g e n e e x p r e s s io n 5 D a y s P 5 B 3 F o ld C h a n g e

Figure 3.5: Gene expression changes of EMT markers induced in P5B3 upon stimulation with TGF-β. The expression of VIM, CDH1, CDH2, FN1, SNAI1, SNAI2, TWIST1 and ZEB1 was compared between untreated and treated P5B3 cells after incubation with 10 ng/ml TGF-β for 5 days. Results were analysed using the comparative ΔΔCT method (Schmittgen, Livak 2008) (n=4). The gene expression was normalised against the TATA-box protein (TBP) gene, which was utilised as reference gene.

87

3.2.1.3 Protein expression changes induced in P5B3 through the treatment with 10 ng/ml TGF-β for 5 days

The molecular changes induced through the treatment of P5B3 indicate the induction of EMT, however, based on the potential variations between gene and protein expression, additional analysis of EMT-associated proteins was performed using immunofluorecense staining. The staining (Fig. 3.6) has shown that P5B3 untreated has a strong expression of CADH1, located at the cell membranes of the cells, whereas no expression of VIME and only low, dispersed FINC expression was detectable at an untreated condition. Upon treatment the expression of CADH1 was strongly reduced and the expression of VIME and FINC strongly increased. The expression of VIME was detected in the cytoplasm, where it comprises, together with the microtubules and microfilaments, the cytoskeleton. Also the expression of FINC was localised in the cytoplasm of the cell. This shows a confirmation of the previously measured molecular changes. The analysis of the EMT- associated proteins confirmed previous findings of the altered gene expression of CDH1,

VIM and FN1 upon stimulation with TGF-β (Fig. 3.5).

3.2.1.4 Quantitative mass spectrometry analysis of untreated and treated P5B3 cell extracts using 10 ng/ml TGF-β for 5 days

In order to investigate proteomic changes through the stimulation with TGF-β, 25 ug of total protein of each growth condition (n=3) was used and label-free quantitative proteomics was performed on the complete cell lysate. The generated library based on all samples contained 1308 different proteins using a 1 % FDR cut-off. Within this library, only 3 EMT markers, CADH1, VIM and FINC, were identified. The comparison of the protein peak areas of treated and untreated samples have shown significant changes in the expression of VIME and FINC, whereas the decrease in the expression of CADH1 was detected, however this decrease did not present a significant difference (Fig. 3.7).

88

Figure 3.6: Representative images of immunofluorescence staining of untreated and treated P5B3 cells after incubation with 10 ng/ml TGF-β for 5 days. The cells were stained for the mesenchymal marker Fibronectin and Vimentin, as well as the epithelial marker E-cadherin. Staining with DAPI is presented as blue and FITC staining represents staining with the marker of interest. The scale bar shows a length of 50 µm

89

Figure 3.7: Comparison of protein peak areas of E-cadherin (CADH1), Vimentin (VIME) and Fibronectin (FINC) for untreated and treated cells of P5B3 using quantitative mass spectrometry analysis (n=3).

For the further analysis, the list of 1308 proteins was reduced through the application of a significance cut-off of 0.05. 365 proteins showed significant differences between the untreated and treated sample groups. Of these 365 proteins, 195 were additionally showing an absolute change of expression of at least 1.5 fold. These 195 proteins were applied to an enrichment analysis using the enrichment tools supplied by the Gene Ontology Consortium (http://www.geneontology.org/ (Accessed 15.03.18). All together, 71 unique proteins were assigned to Gene Ontology terms widely associated with metastasis (Fig. 3.8A). 33 of these were assigned to “cell adhesion”, 24 to “cell migration” and 55 to “tissue development”. Figure 3.8B presents the numbers of unique and shared genes of each of the three selected Gene Ontology terms. Furthermore, their expression directionality and their assigned categories are represented in a heat map (Fig. 3.8C). The terms “cell adhesion”, “cell migration” and “tissue development” were selected due to their involvement in the process of EMT. The analysis using Gene Ontology indicated a succesful alteration of epithelial cells into an increased mesenchymal cell state. An example for protein changes in accordance with the induction EMT are the upregulation of migratory proteins, such as ANXA3 (Annexin 3), and ITAV (Integrin Subunit Alpha V) and the reduced expression of cytoskeletal proteins, such as KRT19 (Keratin 19).

90

Figure 3.8: Analysis of significant proteins (<0.05) with an absolute fold change of 1.5 and higher using Gene Ontology Consortium (http://www.geneontology.org). 71 proteins were assigned to the biological terms of “cell adhesion”, “cell migration” and “tissue development” (A). 12 proteins were detected in all 3 terms (B). The heat map (C) indicates the expression of each protein and the assignment of the proteins to each term. Blue = reduced expression, red = increased expression. The colour coding at the side of the heat map highlights the assigned group (tissue development = purple, cell migration = blue, cell adhesion = green, shared tissue development/cell migration = orange, shared cell migration and cell adhesion = red, shared tissue development/cell adhesion = yellow and detected in all 3 terms = grey).

91

3.2.1.5 Time-point optimisation of treatment length with TGF-β of P5B3 through the analysis of morphological and gene expression changes

The initial results strongly support the use of this cell line model for the discovery of novel biomarkers associated with the process of disease progression in prostate cancer. Based on this, a time point optimisation experiment was performed in which the length of the treatment was optimised and selected. The treatment length was limited to 10 days, based on the minimal required seeding density of the P5B3 cells for healthy cell growth. For the definition of an optimal time point regarding the successful induction of EMT, morphological observations and molecular changes were analysed using bright field microscopy and qRT-PCR on EMT genes and EMT-TFs.

3.2.1.5.1 Morphological changes in P5B3 over time when treated with 10 ng/ml TGF-β

Cells of P5B3 were treated consecutively for 3, 5, 7 and 10 days with 10 ng/ml TGF-β. During this time, the cells were not passaged and kept in one flask throughout the duration of the experiment. This was done to ensure the uninterrupted treatment with TGF-β. Prior to this, a minimum seeding density was defined as 50 000 cells per T175 flask to ensure the healthy growth of the cells (data not shown).

During the time point experiment, the media was changed every second day in both conditions, untreated and treated. The treated media was supplemented with 10 ng/ml TGF-β in each media exchange. The morphological changes in P5B3 across the time points are shown in Figure 3.9. It can be seen that untreated P5B3 do not alter their morphology throughout the growth on tissue culture plastic for 10 consecutive days. Furthermore, the stimulation of P5B3 with TGF-β led, after 3 days, to morphologically visible changes, which increased throughout the stimulation, showing the clearest difference between treated and untreated cells at day 10 (Fig. 3.9). The treated cells have developed an increased elongated cell shape and have shown a separation from the neighbouring cells, whereas the untreated cells retained the “cobblestone” morphology (Fig. 3.9).

92

Figure 3.9: Morphological appearance of untreated and treated cells of P5B3 after growth over 10 days. Brightfield images were taken at the timepoints of 3, 5, 7 and 10 days at a 4x magnification. The scale bar indicates 10 µm.

93

3.2.1.5.2 Gene expression changes in P5B3 over time when treated with 10 ng/ml TGF-β

In addition to the morphological changes observed across the 4 time points, analysis of the gene expression changes of the previously analysed EMT markers; VIM, CDH1,

CDH2, FN1, SNAI1, SNAI2, TWIST1 and ZEB1, was performed across the time points

of 3, 5, 7 and 10 days (Fig. 3.10). The gene expression changes based on the induced fold change of the days 5, 7 and 10 were compared to the fold change induced after the stimulation for 3 days. Vimentin showed an upregulation after 3 days, however a significant stronger increase could be observed after 5 days of stimulation. The vimentin expression at time point 7 still presents a significant increase compared to the time point of 3 days, however less intense when compared to 5 days (Fig. 3.10A). CDH1, the only marker that shows a reduction in its expression is slightly downregulated at the time points 3, 5 and 10, presenting a similar reduction in their expression without any significant differences. The decrease at the time point 7 days presented the strongest and only significant decrease (Fig. 3.10B). CDH2 (Fig. 3.10C), FN1 (Fig. 3.10D), SNAI2 (Fig. 3.10E) and ZEB1 (Fig. 3.10H) have shown a steady increase in the induced gene expression fold change from day 3 to day 10. CDH2 and SNAI2 have presented the strongest fold change increase at day 10, with a more than 150-fold and 6-fold increase in its expression for CDH2 and SNAI2, respectively. The overall analysis highlighted a consistent increase of CDH2 from time point to time point (Fig. 3.10C). Despite the consistent increase of the FN1 expression, no significant differences were detected compared to day 3. It highlights a consistent upregulation of FN1 throughout the length of the stimulation. The expression of the EMT-TF SNAI1 showed strong variation for the days 5 and 7, and therefore only day 10 presented significant increased expression compared to the first induction at day 3 (Fig. 3.10D). ZEB1 presented a consistent 20- fold change across the time point 3, 5 and 7 days and sharply rose to a significant fold change of 60 at day 10. The EMT-TF TWIST1 showed an upregulation of its expression throughout the treatment, with a plateau over 5, 7 and 10 days, however, their overall expression was very low and, for this reason, the fold change analysis showed large variations across repeats and limited significance in their changes was observed (Fig. 3.10G). Significant differences were detected when comparing expressions of 5 and 7 days to 3 days.

Based on the results of this analysis, in which 50 % of the markers showed their strongest upregulation at the time point of 10 days, and the clear morphological changes observed

94

at this time point, 10 days of treatment were selected for the further experimental procedures.

Figure 3.10: Gene expression changes of known EMT markers induced in P5B3 upon stimulation with TGF-β . The gene expression of Vimentin (A), E-cadherin (B), N-cadherin (C), Fibronectin (D), and the EMT-TFs Snail (E), Slug (F), Twist (G) and ZEB1 (H) was measured across four different time points using quantitative real-time PCR and 2-ΔΔCT _{method (Schmittgen, Livak 2008) (n=4). The significance}

analysis was performed comparing the fold change of days 5, 7 and 10 with the fold change difference of each gene induced after treatment for 3 days. The gene expression was normalised against the TATA-box protein (TBP) gene, which was utilised as reference gene.

95

3.2.1.6 Protein expression changes in P5B3 after treatment with 10 ng/ml TGF-β for 10 days using Immunofluorescence staining

To further confirm the changes of expression at time point 10 days, immunofluorescence staining of cells was performed. Cells of both treatment conditions were grown on coverslips placed inside 24-well plates and the immunofluorescence staining was performed inside each well. The staining was performed in triplicate across three separate experiments and representative results are shown in Figure 3.11.

Visible changes in their protein expression were detected for all markers based on the comparison of untreated and treated P5B3 cells. Untreated P5B3 cells did not show VIME expression, and the treatment with TGF-β resulted in an increased expression of VIME visible in the cytoplasm. On the other hand, untreated P5B3 cells have shown a strong expression of the epithelial cell marker CADH1 in the membranes of the cells, which was strongly reduced upon treatment. However, a low protein expression remained detectable in the treated cells, indicating a reduction in the protein expression, but not a complete loss. The third studied marker, FINC, could be detected in single, untreated cells, but a strong increase in its expression was observed through the treatment, resulting in its expression in the majority of the cells (Fig. 3.11). In both conditions, the expression of FINC was associated with the cytoplasm.

96

Figure 3.11: Representative images of immunofluorescence staining of untreated and treated P5B3 cells after treatment for 10 days with 10 ng/ml TGF-β showing the EMT marker E-cadherin, vimentin and fibronectin. Staining with DAPI is presented as blue and FITC staining represents staining with marker of interest. The scale bar shows a length of 50µm.

97

3.2.1.7 Protein expression changes in P5B3 after treatment with 10 ng/ml TGF-β for 10 days using Western blot analysis

As an additional validation of the protein changes induced through the treatment with TGF-β, cell lysates of P5B3 cells in the uninduced and induced cell states were collected and analysed using Western blot analysis (Fig. 3.12). The markers analysed were FINC, VIME, CADH1 and CADH2. Commonly used loading controls are actins or tubulins, which are highly associated with the cytoskeleton. However, during the process of EMT, the cytoskeleton is strongly influenced. To counteract potential bias through this, Cyclophilin A was selected as loading control. In the analysis of the generated sample material, the expression of FINC was consistently upregulated in both biological repeats of the treated compared to the untreated samples. The same was shown for VIME. A reduction in the expression of CADH1 could also be observed, however the intensity of reduction varied between the samples. CADH2 was shown to be upregulated in the treated sample 1 and to a smaller extent also in the treated sample 2, and was not detectable in both untreated samples. Across all samples, the loading control showed a consistent intensity.

Figure 3.12: Western blot of cell lysates generated from untreated and treated P5B3 cells. Protein analysis of the EMT markers Fibronectin, Vimentin, E-cadherin and N-cadherin. Cyclophilin A was used as loading control. 50 µg of protein was loaded for each sample.

98

3.2.2 Development of a second inducible model of