*4.9. Growth Factors xMAP® Technology*

Cytokine concentrations were determined from the non-pigmented lymph node part, using analyte-specific capture beads coated with target-specific capture antibodies according to the manufacturer's specifications. The analytes were detected by biotinylated analytespecific antibodies. Following binding of the fluorescent detection label, the reporter fluorescent signal was measured with the Bio-Plex 200 multiplex suspension array system (Bio-Rad, Hercules, CA, USA) and detected with Bio-Plex 5.0 Software (Bio-Rad, Hercules, CA, USA). The sensitivity for the respective cytokines was as followed: CCL1/I-309 1.73–1260 pg/mL; CCL5/RANTES 8.27–6030 pg/mL; CCL11/Eotaxin 20.23–14,750 pg/mL, CXCL1/GRO alpha 15.30–11,160 pg/mL; IL-8/CXCL8 1.44–1050 pg/mL; CCL2/JE/MCP-1 11.87–8650 pg/mL; CCL8/MCP-2 4.66–3400 pg/mL; CCL17/TARC 30.62–22,320 pg/mL; CXCL10/IP 0.43–310 pg/mL. Analyte levels of complete growth medium mixed with the additives FBS or hPL served as background controls (Supplementary Figure S2).

#### *4.10. DNA Extraction*

Genomic DNA of the primary tumor and all four MUG Mel3 cell lines was isolated on a Maxwell MDxResearch System (Promega).

#### *4.11. Copy Number Profiling*

Genome-wide copy number alterations (CNA) were established using shallow wholegenome sequencing (sWGS). Shotgun libraries were prepared using the TruSeq DNA LT Sample preparation Kit (Illumina, San Diego, CA, USA). Briefly, 380 ng, 144 ng and 360 ng input DNA from all four MUG Mel3 cell lines and both parts of the lymph node were fragmented in 130 µL using the Covaris System (Covaris, Woburn, MA, USA). After concentrating the volume to 50 µL, end repair, A-tailing and adapter ligation were performed following the manufacturer's instructions. For selective amplification of the library fragments that have adapter molecules on both ends, 15 PCR cycles were used for higher concentration samples. Libraries were quality checked on an Agilent Bioanalyzer using a DNA 7500 Chip (Agilent Technologies, Santa Clara, CA, USA) and quantified using qPCR with a commercially available PhiX library (Illumina, San Diego, CA, USA) as a standard. Libraries were pooled equimolarily and sequenced on an Illumina MiSeq in a 150 bp single read run. On completion of the run data were base-call demultiplexed on the instrument (provided as Illumina). Genome-wide copy number calling was performed as previously described [50].

#### *4.12. Mutation Analysis Using AmpliSeq (BRAF)*

Highly multiplexed PCR was used to generate amplicon libraries to amplify 207 amplicons, covering approximately 2800 COSMIC mutations from 50 oncogenes and tumor suppressor genes. (Cancer Hotspot Panel v2, Cat. Nr. 4475346; Thermo Fisher Scientific, Waltham, MA, USA). All analyses were performed in duplicate. Libraries were prepared using the Ion AmpliSeq Library Kit 2.0 and sequencing was performed on an Ion Proton Sequencer (Thermo Fisher Scientific, Waltham, MA, USA). Emulsion PCR and sequencing runs were performed with the appropriate kits (Ion One Touch Template Kit version 2 and Ion Proton 200 Sequencing Kit; Thermo Fisher Scientific) using Ion PI chips. Sequencing length was set to 520 flows and yielded reads ranging from 70 to 150 bp, consistent with the expected amplicon size range. Initial data analysis was performed using the Ion Torrent Suite Software version 4.1 Plug-ins (Thermo Fisher Scientific).

#### *4.13. Tumorigenicity Study*

CR ATH HO mice (Crl:NU(NCr)-Foxn1nu, Charles River Laboratories, Kent, UK) and NXG mice (NOD-Prkdcscid-IL2rgTm1/Rj, Janvier Labs, Saint Berthevin Cedex, France) were maintained in-house (4–5 weeks of age, weight between 15 and 20 g). In accordance with a protocol approved by the committee for institutional animal care and use at the Austrian Federal Ministry of Science and Research (BMWFW); vote 66.010/0046- WF/V/3b/2016) animal work was carefully carried out. All mice were maintained under specific pathogen free (SPF) conditions in individually ventilated cages with ad libitum access to food and water. For cell injection and ultrasound imaging, mice were anaesthetized with constant administration of 2% isoflurane in a constant airflow of 2.5 L per minute. Animals were sacrificed 25 days post-injection.

Mice were divided into two groups (*n* = 5) according to strain. Animals were injected into each flank subcutaneously with 2.5 <sup>×</sup> <sup>10</sup><sup>6</sup> cells in 100 µL PBS cell suspension using a 27G needle. Injection was performed as described in the following: front right flank received MUG Mel3 NPh, back right flank received MUG Mel3 NPF, front left flank received MUG Mel3 Ph, back left flank received MUG Mel3 PF. Inoculation of the tumor was monitored daily and ultrasound imaging started on day six post-injection once a week. After sacrifice, histopathological examination was performed. The mice were dissected, and tumors extracted. All other organs were checked visually for structural changes. The

tissue was fixed in 4% paraformaldehyde solution for 24 h, and were further embedded in paraffin.

#### *4.14. Ultrasound Imaging*

High-frequency ultrasound (HF-US) was performed using a Vevo3100 HF-US system with a 50 MHz (MX700) or a 40 MHz (MX550D) transducer (Fujifilm VisualSonics, Inc., Toronto, ON, Canada) reaching a spatial resolution of about 30–40 µm. Images in sagittal and transverse planes were obtained of the region of interest. Calculation of tumor volume was performed using VevoLab Software (Version 5.5.1, Fujifilm VisualSonics, Inc.) using the formula displayed in Equation (1):

$$\text{V}\left[\text{mm}^3\right] = \frac{\text{3.141} \times \text{length}\left[\text{mm}\right] \times \text{width}\left[\text{mm}\right] \times \text{depth}\left[\text{mm}\right]}{6} \tag{1}$$

#### *4.15. PS Exposure*

PS exposure was measured using the RealTime-Glo™ Annexin V Apoptosis Assay (Promega). Briefly, cells were seeded at 10<sup>5</sup> cells/100 µL medium in a white 96-multiwell plate with clear bottom and incubated O/N at 5% CO<sup>2</sup> and 37 ◦C. The reagent stock solution (1000×) was diluted in respective medium according to the manufacturer's protocol. Before the measurement 100 µL of the reagent solution was added to the cell suspension. Luminescence was measured using the Glomax Multi+ detection system (Promega, Madison, WI, USA).

#### *4.16. Peptides*

C-terminally amidated peptides R-DIM-P-LF11-322 (PFWRIRIRRPRRIRIRWFP-NH2, M = 2677.4 g/mol) and R-DIM-P-LF11-334 (PWRIRIRRPRRIRIRWP-NH2, M = 2382.4 g/mol) derived from Lactoferricin were purchased from PolyPeptide Group (San Diego, CA, USA). A purity of higher than 96% for all peptides had been determined by RP-HPLC. Peptide stock solutions were prepared in acetic acid (0.1%, *v*/*v*) to an approximate concentration of 3 mg/mL and treated by ultrasonication at 15 min for better solubility. Peptide concentration was determined by measurement of UV absorbance of tryptophan at a wavelength of 280 nm using a NanoDrop photometer (ND 1000, Peqlab, VWR International, Inc., Erlangen, Germany). All peptide stocks were stored at 4 ◦C until use.

#### *4.17. PI Uptake Toxicity Assay*

To determine peptide-induced cell death, cells were collected, resuspended in respective media and diluted to a concentration of 1 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/mL. 100 µL aliquots (10<sup>5</sup> cells) were incubated with different peptide concentrations (5 µM to 100 µM) for up to 8 h in the presence of propidium iodide (PI; 2 µL/10<sup>5</sup> cells of 50 µg/mL, Molecular Probes Inc., Eugene, OR, USA) at RT in black 96-well plates. PI uptake was measured using the GloMax® Multi+ Detection System (Promega, Madison, WI, USA). Cytotoxicity was calculated from the percentage of PI-positive cells in media alone (P0) and in the presence of peptide (PX; Equation (2)). Triton-X-100 was used to determine 100% of PI positive cells (P100).

$$\% \text{ PI} - \text{uptake} = \frac{100 \times (\text{P}\_{\text{x}} - \text{P}\_{0})}{(\text{P}\_{100} - \text{P}\_{0})} \tag{2}$$

Excitation and emission wavelengths were 536 nm and 617 nm, respectively. Each experiment was repeated at least 3 times.

#### *4.18. Statistical Analysis*

Statistical analyses were conducted in GraphPad Prism 9.2.0 (GraphPad Software, San Diego, CA, USA) using two-tailed student's *t*-tests to compare culture conditions (FBS or hPL) within cell line origins (pigmented or non-pigmented) or to compare the origins of cells within the same cultivation method, and two-tailed unpaired student's *t*-test to

compare tumor weight and tumor growth of the four culture conditions on the day of sacrifice. A *p*-value ≤ 0.05 was considered statistically significant.

#### **5. Conclusions**

Melanoma in pregnancy is a vast, debated and complex field. Further research is urgently needed to provide evidence-based treatment and improve the management of melanoma in pregnancy. We have successfully established four cell lines from a melanoma metastasis from a pregnant woman. By using different tumor fractions and cultivation conditions, we were able to better mimic intratumoral heterogeneity as the four cell lines differ morphologically, genetically and phenotypically. In addition, we were able to demonstrate different cytotoxic effects by antitumor peptide treatments for all four cell lines, and thus may also consider them for new innovative treatment strategies.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ijms222111318/s1.

**Author Contributions:** B.R. and S.S. designed the draft for this manuscript. S.S. performed main cell culture parts, Luminex Technology, collected and analyzed the data and wrote the manuscript with B.R. T.H. performed qPCR, S.A.W. and I.A. conducted the animal experiments. S.R. and D.Z. were responsible for the peptides. E.R. was responsible for the patient and patient history and acquired together with G.R. the tumor material. C.B. and P.S. prepared and supplied the hPL. A.A. provided the human specimen and was responsible for IHC stainings. W.B., B.H. and C.W. performed ICC stainings. M.G. and N.K. contributed to in vitro experiments. K.K. and E.H. were responsible for the genetic section of this manuscript. G.R. helped organizing the study. B.R. managed, supervised, and provided funding for this study. All authors contributed to the interpretation of the data, discussed the results, contributed to the manuscript, and approved the final version. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We thank the DocSchool 'Molecular Medicine and Inflammation' at the Medical University of Graz for funding the publication costs.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

