*4.6. Proteomics*

#### 4.6.1. Sample Preparation

Approx. 10<sup>6</sup> cell pellets (*n* = 1 technical replicate per condition deriving from *n* = 3 to 4 biological replicate samples) were resuspended in 50 μL PBS following the addition of 50 μL 1% SDS in 100-mM Hepes/NaOH, pH 8.5 supplemented with protease inhibitor cocktail EDTA-free (11836170001, Sigma-Aldrich, Buchs, Switzerland). Samples were heated to 95 ◦C for 5 min, transferred on ice, and benzonase (71206-3, Merck AG, Zug, Switzerland) was added to degrade DNA at 37 ◦C for 30 min. Samples were reduced by the addition of 2 μL of a 200-mM DTT solution in 200-mM Hepes/NaOH, pH 8.5 and, subsequently, alkylated by the addition of 4 μL of a 400-mM chloroacetamide (CAA, #C0267, Sigma-Aldrich, Buchs, Switzerland) solution in 200 mM Hepes/NaOH, pH 8.5. Samples were incubated at 56 ◦C for 30 min. Access CAA was quenched by the addition of 4 μl of a 200-mM DTT solution in 200 mM Hepes/NaOH, pH 8.5. Lysate were subjected to an in-solution tryptic digest using the single-pot solid phase-enhanced sample preparation (SP3) protocol [59,60]. To this end, 20 μL of Sera-Mag Beads (#4515-2105-050250 and 6515-2105-050250, Thermo Fisher Scientific, Basel, Switzerland) were mixed, washed with H2O and resuspended in 100 μL H2O. Two microliters of freshly prepared bead mix and 5 μl of an aqueous 10% formic acid were added to 40 μL of lysates to achieve an acidic pH. Forty-seven microliters of acetonitrile were added, and samples were incubated for 8 min at room temperature. Beads were captured on a magnetic rack and washed three times with 70% ethanol and once with acetonitrile. Sequencing grade-modified trypsin (0.8 μg; V5111, Promega AG, Dübendorf, Switzerland) in 10 μL 50 mM Hepes/NaOH, pH 8.5 were added. Samples were digested overnight at 37 ◦C. Beads were captured and the supernatant transferred and dried down. Peptides were reconstituted in 10 μL of H2O and reacted with 80 μg of TMT10plex (#90111, Thermo Fisher Scientific, Basel, Switzerland) [61] label reagent dissolved in 4 μL of acetonitrile for 1 h at room temperature. Excess TMT reagent was quenched by the addition of 4 μL of an aqueous solution of 5% hydroxylamine (438227, Sigma-Aldrich, Buchs, Switzerland). Mixed peptides were subjected to a reverse-phase clean-up step (OASIS HLB 96-well μElution Plate, 186001828BA, Waters Corporation, Milford, MA, USA) and analyzed by LC-MS/MS on a Q Exactive Plus (Thermo Fisher Scientific, Basel, Switzerland), as previously described [62].

#### 4.6.2. Mass Spectrometric Analysis

Briefly, peptides were separated using an UltiMate 3000 RSLC (Thermo Scientific, Basel, Switzerland) equipped with a trapping cartridge (Precolumn; C18 PepMap 100, 5 Lm, 300 Lm i.d. × 5 mm, 100 A◦) and an analytical column (Waters nanoEase HSS C18 T3, 75 Lm × 25 cm, 1.8 Lm, 100 A◦). Solvent A: aqueous 0.1% formic acid and Solvent B: 0.1% formic acid in acetonitrile (all solvents were of LC-MS grade). Peptides were loaded on the trapping cartridge using solvent A for 3 min with a flow of 30 μL/min. Peptides were separated on the analytical column with a constant flow of 0.3 μL/min applying a 2 h gradient of 2–28% of solvent B in A, followed by an increase to 40% B. Peptides were directly analyzed in positive ion mode, applied with a spray voltage of 2.3 kV and a capillary temperature of 320◦C using a Nanospray-Flex ion source and a Pico-Tip Emitter 360 Lm OD × 20 Lm ID;, 10 Lm tip (New Objective, Littleton, MA, USA). MS spectra with a mass range of 375–1.200 m/z were acquired in profile mode using a resolution of 70,000 (maximum fill time of 250 ms or a maximum of 3 <sup>×</sup> 106 ions (automatic gain control, AGC)). Fragmentation was triggered for the top 10 peaks with 2–4 charges on the MS scan (data-dependent acquisition), with a 30 s dynamic exclusion window (normalized collision energy was 32). Precursors were isolated with a 0.7 m/z window and MS/MS spectra were acquired in profile mode with a resolution of 35,000 (maximum fill time of 120 ms or an AGC target of 2 <sup>×</sup> <sup>10</sup><sup>5</sup> ions).

#### 4.6.3. Raw MS Data Analysis

Acquired data were analyzed using IsobarQuant [63] and Mascot V2.4 (Matrix Science, Chicago, IL, USA) using either a reverse-UniProt FASTA Mus musculus (UP000000589) or Homo sapiens (UP000005640) database. Moreover, a combined database thereof was generated and used for the analysis. These databases also included common contaminants. The following modifications were taken into account: Carbamidomethyl (C, fixed), TMT10plex (K, fixed), Acetyl (N-term, variable), Oxidation (M, variable) and TMT10plex (N-term, variable). The mass error tolerance for full-scan MS spectra was set to 10 ppm and for MS/MS spectra to 0.02 Da. A maximum of 2 missed cleavages were allowed. A minimum of 2 unique peptides with a peptide length of at least seven amino acids and a false discovery rate below 0.01 were required on the peptide and protein levels [64].

#### 4.6.4. MS Data Analysis

The raw output files of IsobarQuant (protein.txt files) were processed using the R programming language (ISBN 3-900051-07-0). As a quality filter, only proteins were allowed that you were quantified with at least two unique peptides. Human and mouse samples were searched against a combined human and mouse database and annotated as unique for human or mouse or mixed. Raw signal-sums (signal\_sum columns) were normalized using vsn (variance stabilization normalization) [65]. In order to try to annotate each observed ratio with a *p*-value, each ratio distribution was analyzed with the locfdr function of the locfdr package [66] to extract the average and the standard deviation (using the maximum likelihood estimation). Then, the ratio distribution was transformed into a z-distribution by normalizing it by its standard deviation and mean. This z-distribution was analyzed with the fdrtool function of the fdrtool package [67] in order to extract *p*-values and false discovery rates (fdr, *q*-values).

#### *4.7. Tissue Microarray*

Tissue microarray core annotations and quantification of positive staining were performed by QuPath software version v0.2.0-m8 [68] using the TMA map function. Kaplan–Meier curves to calculate the association between TNC-positive cells and disease progression were calculated using the "survfit" function and the global Log-Rank test using the Survival R package [69,70]. To estimate the survival, we used the function "surv\_cutpoint", which employs maximally selected rank statistics (maxstat) to determine the optimal cut-point for continuous variables [18]. For pairwise comparison, the *p*-value was estimated by the Log-Rank test and adjusted with the Benjamini–Hochberg (BH) method. If no information on patient outcome was available, information at the last follow-up was used for all parameters. Clinical progression was defined as metastasis or local recurrence. Disease progression was defined by combining any form of recurrence (PSA and clinical progression). Data representation and graphical plots were generated using the ggplot2 R package [71]. Data analyses were done using RStudio version 1.1.463 [72] and R version 3.5.3 [73].

#### *4.8. Immunohistochemistry*

FFPE sections (4 μm) were deparaffinized and used for heat-mediated antigen retrieval (citrate buffer, pH 6, Vector Labs). Sections were blocked for 10 min in 3% H2O2, followed by 30 min, RT incubation in 1% BSA in PBS–0.1%Tween 20. The following primary antibodies were used (Table 3):


**Table 3.** Primary antibodies used for Immunohistochemistry

Secondary anti-rabbit antibody Envision HRP (DAKO, Agilent Technologies, Basel, Switzerland) for 30 min or anti-rat HRP (Thermo Scientific, Basel, Switzerland). Signal detection with AEC substrate

(DAKO, Agilent Technologies, Basel, Switzerland). Sections were counterstained with Hematoxylin and mounted with Aquatex.

#### *4.9. Immunofluorescence*

After deparaffinization, heat-mediated antigen retrieval (citrate buffer, pH 6, Vector Labs) was performed. Sections were blocked in 1% BSA in PBS–0.1% Tween 20 for 30 min, RT incubation. The primary antibodies used (Table 4), were incubated overnight in blocking solution at 4 ◦C:


**Table 4.** Primary antibodies used for Immunofluorescence

Secondary anti-rabbit/mouse/goat/rat antibodies coupled to Alexa Fluor®-488, 555 or 647 fluorochrome conjugates (Invitrogen, Thermo Scientific, Basel, Switzerland) were incubated for 90 min at 1:250 dilution in PBS. Sections were counterstained with DAPI solution (Thermo Scientific, Basel, Switzerland, final concentration 1 μg/mL in PBS, 10 min), washed and mounted with prolonged diamond antifade reagent (Invitrogen, Thermo Scientific, Basel, Switzerland).
