Cell culture and maintenance

Equipment and instruments with product name, catalogue number and producer are listed in Appendix B, Supplementary Table 7.

3.1.1 Cell lines

The murine melanoma cell line B16-F10 derived from the skin of the albino mice strain

C57BL/6N was used in experiments to evaluate MITF and phenotype switching upon tankyrase inhibitor treatment. To investigate β-catenin-mediated immune evasion and whether WNT signaling affects MITF expression in B16-F10, two β-catenin knockout cell lines were produced by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based knockout (KO). B16-F10^Ctnnb1KO was designed using CHOPCHOP v2, a web tool of CRISPR genome engineering [25, 93]. The knockout sequence from wild-type B16-F10 was 5′-

GATTAACTATCAGGATGACG-3′, and by amplifying this gene fragment in a polymer chain reaction (PCR), we could verify a successfully performed knockout. Immunofluorescence with β-catenin was also performed for additional verification [25].

The effect of tankyrase inhibitors on WNT and YAP signaling depends on cell lineage and context [25]. Previous studies have shown synergistic anti-PD-1/tankyrase inhibitor treatment effects in B16-F10 tumors [25]. However, genetic background and cell signaling pathways may cause variable results between different melanomas [25]. Furthermore, the B16-F10 murine mouse model only partially recapitulates human melanoma. Therefore, a panel of human melanoma cell lines shared by Dr. Eivind Hovig (Oslo University Hospital-Radiumhospitalet) was utilized further to investigate the effects of tankyrase inhibitors in vitro. This master thesis used the following human cell lines listed in Table 1 to analyze transcriptional and protein level regulation.

29 Table 1. The panel of human melanoma cells

Human melanoma cell lines

3.1.2 Cultivation of cells

All cell lines were cultured in cell culture flasks at 37 °C in 5% CO2. Cells were cultured in Roswell Park Memorial Institute – 1640 medium (RPMI) with 5% fetal bovine serum (FBS), providing amino acid and growth factors. To prevent bacterial contamination, 1%

penicillin/streptomycin solution (Pen-strep) was added to all cell culture mediums.

3.1.3 Cell splitting and passaging

All cell cultures were inspected daily using a microscope to keep cells in an exponential growth phase and a maximum confluence below 80-90%, before passaging the cells in a 1:10 split ratio about twice a week. After removing the used cell culture medium, the cells were washed once using phosphate-buffered saline (PBS) and split with trypsin/EDTA solution detaching the cells from the plastic surface of cell culture flasks. The flasks were next incubated for ~10 minutes at 37 °C until the cells were visibly detached. The addition of new medium containing FBS leads to the inactivation of trypsin. An increasing passaging number was noted on the flask for each cell culture passage, and all cell cultures were kept below 20 passages. All work with cell culture was performed in sterile environments.

3.1.4 Cell seeding

Cells were seeded one day before treatment in an appropriate amount to reach ~80-90%

confluence after 72 hours of treatment.10 µl of detached cell suspension sample were inserted into each chamber on Bio-Rad counting slides for determining cell concentration before seeding.

The slides were inserted into a TC20™ automated cell counter. After determining cell

30 concentration, an appropriate amount of cells were calculated and seeded depending on the concentration and size of the seeding plate ratio (Table 2).

Table 2. Plate seeding ratio of cells Plate Cell number

Aliquots cell cultures, with low passage numbers, were stored in liquid nitrogen at -196ºC for long-term storage. After detaching the cells with trypsin, the cells were resuspended in 1 ml medium containing 10% Dimethyl Sulfoxide (DMSO), a cryoprotective agent, and transferred to a CryoTube. The aliquots were transferred to a Mr.Frosty™ Freezing container, with 100%

isopropyl alcohol and kept at -80°C. Storage of Mr.Frosty overnight at -80°C leads to a slow cooling rate at about 1°C/min. CryoTubes were transferred to liquid nitrogen the next day for optimal preservation.

3.1.6 Cell thawing

Frozen cells were taken from the nitrogen tank and thawed in a 37°C water bath for about 1 minute. 1 ml of cells were transferred to 15 ml Falcon tubes and centrifuged at 2000 rounds per minute (rpm) for 3 minutes. After removing the supernatant, the remaining pellet was

resuspended in 1 ml pre-warmed cell culture medium and transferred to a cell culture flask.

3.1.7 Cell line authentication and mycoplasma detection

Mycoplasma testing of all cell cultures was performed monthly, to ensure the absence of mycoplasma contaminations, using a MycoAlert™Mycoplasma Detection Kit. The kit detects common contaminants through luciferase enzymes in the Mycoalert TM substrate. Cell cultures infected with mycoplasma can acquire abnormal cell physiology, metabolism, and cell growth.

Further effects of mycoplasma contamination are chromosomal aberrations, change of gene expression patterns, cell death, and changes in cell membrane antigenicity. Significant sources of mycoplasma contamination in cell cultures are medium, improper sterilization, laboratory

personnel, and incubation [94]. Two chambers were used to minimize mycoplasma spreading in the 5% CO2 chamber used for cell incubation. One was mycoplasma-free cultures (tested), and the other was cultures yet to be tested (quarantine). All cultures used in this study were free of mycoplasma.

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3.1.8 Drugs and substances

The first tankyrase inhibitors discovered for repressing WNT signaling were XAV939, and IWR-1 [95]. Specific tankyrase inhibitors such as G007-LK were later discovered, a hydrophobic small-molecule that dose-dependently inhibits cell growth. G007-LK suppresses WINT signaling by blocking the PARsylation activity of tankyrase (see 1.5 Tankyrase) and suppressing YAP signaling by upregulating AMOT proteins (see 1.5.4 Tankyrase inhibition suppress YAP/TAZ activity). In our lab, G007-LK (molecular weight of 529,96) was dissolved in DMSO, resulting in a 10 mM stock solution, and stored at -4°C. For in vitro experiments, the stock was further diluted to 10 nM in a cell culture medium.

WNT3a is one of 19 members of WNT ligands, which activates the WNT signaling pathway [96]. In our studies, recombinant WNT3a was used. WNT3a was diluted with 0.1% BSA.

Combinational treatment with WNT3a and G007-LK was used to investigate if G007-LK could counteract the WNT signaling activating effect of WNT3a.