Tight Junction Protein Claudin-12 Is Involved in Cell Migration During Metastasis

biomolecules

Article Tight Junction Protein Claudin-12 Is Involved in Cell Migration during Metastasis

Desislava Kolchakova, Dzhemal Moten , Tsvetelina Batsalova and Balik Dzhambazov *

Department of Developmental Biology, Plovdiv University “Paisii Hilendarski”, 4000 Plovdiv, Bulgaria; [email protected] (D.K.); [email protected] (D.M.); [email protected] (T.B.) * Correspondence: [email protected]; Tel.: +359-32-261-535

Abstract: Claudins are important components of the tight junctions determining barrier properties, cell polarity, and paracellular permeability. Although many functions of claudins in cancer cells have not been elucidated, recent studies have shown that claudins play an important role in cell migration and metastasis. Loss of epithelial/endothelial integrity, disruption of tight junctions, and increased paracellular leakage are often observed during metastasis. The aim of our study was to investigate the involvement of claudin-12 in the process of cell migration as well as to evaluate the possibility of using this protein as a speciﬁc target for the regulation of tumorigenesis. We have performed immunocytochemistry assays to detect the expression of claudin-12 in different epithelial/endothelial human cell lines, and selected three (A549, LS180, and HeLa) for further experiments. Using transwell chamber migration assays, we found that anti-claudin-12 antibodies inhibited both the migration and proliferation of claudin-12 expressing cells (A549 and LS180), inducing apoptosis, as well as the migration capacity of Jurkat cells through the monolayers formed

from A549 or LS180 cells. In addition, co-cultures of Jurkat cells on monolayers from A549 or LS180 cells, in the presence of synthetic claudin-12 peptides representing the extracellular domains

Citation: Kolchakova, D.; Moten, D.; of the claudin-12 protein, also reduced the number of migrated Jurkat cells. Two of the tested Batsalova, T.; Dzhambazov, B. Tight peptides (p5 and p6) almost completely blocked the migration of Jurkat cells. All migrated Jurkat Junction Protein Claudin-12 Is cells expressed LFA-1 and CD62L, but not CD44. Thus, claudin-12 is a suitable biomarker for tumor Involved in Cell Migration during progression and metastasis and an attractive target for antitumor therapy. Anti-claudin-12 antibodies Metastasis. Biomolecules 2021, 11, 636. and competitive inhibitory peptides could be useful in the therapeutic approach applied to cancer https://doi.org/10.3390/biom metastasis in tissues expressing claudin-12. 11050636 Keywords: claudin-12; cell migration; antibody treatment; peptide inhibition; metastasis; tight junctions Academic Editor: Claudia Mierke

Received: 1 April 2021 Accepted: 23 April 2021 1. Introduction Published: 25 April 2021 Claudins are small (20–34 kDa) tetraspanning transmembrane proteins that are in-

Publisher’s Note: MDPI stays neutral volved in the structure of tight junctions [1,2]. They play a critical role in the regulation with regard to jurisdictional claims in of paracellular permeability to ions and small molecules in endothelia or epithelia and published maps and institutional afﬁl- the maintenance of cell polarity [3,4]. In addition, claudins are associated with various iations. signaling pathways related to cell proliferation and differentiation [5,6]. The mammalian claudin gene family consists of 27 members, as their expression levels and subcellular localization depend on the tissue and cell type [7,8]. Among claudin proteins, claudin-12 is deﬁned as an unusual member because it does not possess an intracellular PDZ binding motif [1,9], which mediates the interaction with the cytoskeleton. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Claudin-12 is expressed in epithelia and endothelia of the gastrointestinal tract, inner ear, This article is an open access article and brain endothelial cells [1,8,10], as well as in the smooth and striated muscle cells, distributed under the terms and neurons, and astrocytes [11]. It has been shown that in vitro expression of claudin-12 is 2+ conditions of the Creative Commons up-regulated by vitamin D, suggesting an essential role for this claudin in Ca absorption Attribution (CC BY) license (https:// between intestinal epithelial cells [12]. creativecommons.org/licenses/by/ Dysregulated expression of claudins has been reported in various cancers, suggesting 4.0/). that they may have an important role in the migration, invasion, and metastasis of cancer

Biomolecules 2021, 11, 636. https://doi.org/10.3390/biom11050636 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 636 2 of 13

cells [3,13–17]. More than 90% of cancer-related deaths are due to metastases [18]. The metastatic process requires transendothelial migration of the cancer cells. They have to leave their primary site by intravasation in the lumen of the vasculature, to circulate in the bloodstream, and to extravasate to a secondary site [18,19]. Metastasis is accompanied by disruption of tight junctions, loss of epithelial/endothelial integrity, and increased paracellular leakage, providing a space for the mobility of the cancer cells [20]. It was reported that overexpression of claudin-12 signiﬁcantly increased the metastatic properties of human bronchial epithelial cells BEAS-2B [17]. However, the role of claudins in migration of cancer cells through the tight junctions during metastasis is not fully understood. The aim of this study was to investigate the involvement of tight junction protein claudin-12 in the process of metastasis as well as to evaluate the possibility of using this protein as a speciﬁc target for the regulation of tumorigenesis. We hypothesized that cancer cells use claudin-12 to migrate through the tight junctions during metastasis and that blocking this protein or the competitive binding of cancer cells to peptides derived from the extracellular part of claudin-12 will reduce the metastatic process.

2. Materials and Methods 2.1. Cell Lines and Culture Conditions The human cell lines A549 (epithelial lung carcinoma cells, ATCC CCL 185, NBIMCC 2404), Caco-2 (epithelial colon adenocarcinoma cells, ECACC 86010202), HT-29 (epithelial colon adenocarcinoma cells, ECACC 91072201), LS180 (Dukes’ type B epithelial colon adenocarcinoma, mucin-secreting cells, ECACC 87021202), SK-Hep-1 (endothelial liver adenocarcinoma cells, ATCC HTB 52, NBIMCC 1858), HeLa (epithelial cervix adenocarcinoma cells, ATCC CCL 2, NBIMCC 164) and Jurkat E6.1 (acute lymphoblastic leukemia T cells, ECACC 88042803) were obtained either from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, United Kingdom) or from the National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC). All adherent cell lines were cultured in complete medium (CM) containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and stabilized antibiotic ◦ antimycotic solution (all from Merck KGaA, Darmstadt, Germany) at 37 C, 5% CO2, 95% atmospheric air in a humidified incubator. The Jurkat cells were maintained at the same conditions using RPMI 1640 medium (Merck KGaA, Darmstadt, Germany) instead of DMEM. Prior to the performed assays, the cell lines were expanded in 75 cm2 culture flasks (TPP, Trasadingen, Switzerland).

2.2. Reagents and Antibodies All reagents were of analytical quality and obtained from Merck KGaA (Darmstadt, Ger- many), unless otherwise stated. Six synthetic peptides (p1-peptide, YNIHLNKKFEPVFSFDYA, Cldn-12 (157-174), second extracellular loop; p2-peptide, NWRKLRLITFNRNEKNLTVY Cldn-12 (32-51); p3-peptide, TGLWVKCARYDGSSDCLMYD Cldn-12 (52-71); p4-peptide, TTWYSSVDQLDLRVLQ Cldn-12 (72–87); p5-peptide, NRNEKNLTVYTGLWVKCARY Cldn- 12 (42–61); and p6-peptide, DGSSDCLMYDTTWYSSVDQL Cldn-12 (62-81), parts of the first extracellular loop) representing sequences from the extracellular domains of the protein claudin-12 (Figure1) were synthesized from Schafer-N (Copenhagen, Denmark). Monoclonal anti-CLDN12 antibody produced in mouse (clone 2D8, cat. No SAB1403012), anti-mouse IgG (whole molecule)–FITC antibody produced in goat (cat. No F0257), and anti-mouse IgG (whole molecule)–Peroxidase antibody produced in goat (cat. No A4416) were purchased from Merck KGaA (Darmstadt, Germany). Monoclonal anti-human CD11a/CD18-APC (LFA-1) antibody (clone m24, cat. No 363410), anti-human CD62L-FITC (L-selectin) antibody (clone DREG-56, cat. No 304804), and anti-human CD44-PE antibody (clone BJ18, cat. No 338808), all produced in mouse, were purchased from BioLegend® (San Diego, CA, USA). Annexin V-FITC and propidium iodide (PI) were purchased from Abcam (Cambridge, UK). Biomolecules 2021, 11, x FOR PEER REVIEW 3 of 13

from Merck KGaA (Darmstadt, Germany). Monoclonal anti-human CD11a/CD18-APC (LFA-1) antibody (clone m24, cat. No 363410), anti-human CD62L-FITC (L-selectin) antibody (clone DREG-56, cat. No 304804), and anti-human CD44-PE antibody (clone BJ18, Biomolecules 2021, 11, 636 cat. No 338808), all produced in mouse, were purchased from BioLegend® (San Diego, 3CA, of 13 USA). Annexin V-FITC and propidium iodide (PI) were purchased from Abcam (Cam- bridge, UK).

FigureFigure 1. 1. ModelModel of of claudin-12 claudin-12 protein protein showing showing the the extr extracellularacellular domains domains (loop-1 (loop-1 and and loop-2) loop-2) and and designed peptides. Peptide 1 (p1) represents the sequence of loop-2/red, CLDN12 (157–174). Pep- designed peptides. Peptide 1 (p1) represents the sequence of loop-2/red, CLDN12 (157–174). Peptides tides p2, p3, p4, p5, and p6 represent the sequence of loop-1/blue, CLDN12 (32–87). p2, p3, p4, p5, and p6 represent the sequence of loop-1/blue, CLDN12 (32–87).

2.3.2.3. Immunocytochemistry Immunocytochemistry and and Western Western Blotting Blotting A549,A549, Caco-2, HT-29,HT-29, LS180, LS180, SK-Hep-1 SK-Hep-1 and HeLaand HeLa cells werecells seeded were (1.0seeded× 10 5(1.0cells/well) × 105 cells/well)on coverslips on coverslips in 12-well in plates 12-well (TPP, plates Trasadingen, (TPP, Trasadingen, Switzerland) Switzerland) and cultured and in cultured a humid- ◦ inified a humidified incubator atincubator 37 C, 5% at CO37 °C,2 in 5% complete CO2 in DMEM complete (1 mL/well).DMEM (1 mL/well). After 48 h After incubation 48 h incubationperiod, the period, cells were the cells washed were twicewashed with twic Dulbecco’se with Dulbecco’s phosphate phosphate buffered buffered saline (DPBS)saline (DPBS)and fixed and with fixed ice with cold ice acetone:methanol cold acetone:methanol (1:1). (1:1). Then, Then, cells werecells were stained stained for 30 for min 30 min with withanti-CLDN12 anti-CLDN12 antibody antibody at room at room temperature, temperat washedure, washed with DPBS,with DPBS, and incubated and incubated for 15 minfor 15with min secondary with secondary anti-mouse anti-mouse IgG–FITC IgG–FITC antibodies antibodies in darkness. in darkness. After washing After 3washing times with 3 timesDPBS, with the DPBS, expression the expression of claudin-12 of claudin-12 in the studied in the cell studied lines was cell observed lines was using observed a fluorescent using amicroscope fluorescent (Leicamicroscope Microsystems (Leica Microsyste GmbH, Wetzlar,ms GmbH, Germany). Wetzlar, For Germany). the next experiments, For the next we experiments,selected two we claudin-12 selected positivetwo claudin-12 cell lines po (A549sitive andcell LS180)lines (A549 and oneand negativeLS180) and (HeLa). one negative (HeLa).For Western blot analysis of claudin-12, cells (A549, LS180, and HeLa) were washed twiceFor in Western cold DPBS blot and analysis lysed of with claudin-12, RIPA lysis cells buffer (A549, (150 LS180, mM and NaCl, HeLa) 0.5 mM were EDTA, washed 1% twiceTriton in X-100, cold DPBS 0.1% and sodium lysed dodecyl with RIPA sulfate, lysis 50 buffer mM Tris-HCl,(150 mM pHNaCl, 8.0) 0.5 containing mM EDTA, a cOm- 1% Tritonplete™ X-100,protease 0.1% inhibitor sodium dodecyl cocktail sulfate, (Merck 50 KGaA, mM Tris-HCl, Darmstadt, pH Germany). 8.0) containing Lysates a cOm- were plete™sonicated protease for 20 inhibitor s and centrifuged cocktail (Merck at 6000 KGaA,× g for Darmstadt, 20 min. The Germany). supernatants Lysates were were then ® sonicatedfiltered and for concentrated20 s and centrifuged by using at 30 6000× kDa and g for 10 20 kDa min. Amicon The supernatantsUltra-15 Centrifugal were then Filter fil- teredUnits and (Millipore). concentrated Protein by concentrationsusing 30 kDa and were 10 measured kDa Amicon on a® NanoDrop Ultra-15 Centrifugal 2000 UV-Vis Filter spec- Unitstrophotometer (Millipore). (Thermo Protein Fisher concentrations Scientific, Wilmington,were measured DE, USA).on a NanoDrop Equal amounts 2000 (30 UV-Visµg) of spectrophotometerproteins (between (Thermo 10–30 kDa) Fisher from Scientific the 3 cell, Wilmington, lines were subjected DE, USA). to non-heat-denaturedEqual amounts (30 μproteing) of proteins gel electrophoresis (between 10–30 (4–20% kDa) polyacrylamide from the 3 cell gradient lines were ready subjected mini-gels, to non-heat-de- Bio-Rad Labo- naturedratories, protein Hercules, gel electropho CA). Separatedresis (4–20% fractions polyacrylamide were electrotransferred gradient ready onto amini-gels, nitrocellulose Bio- membrane followed by blocking with 5% nonfat dry milk in DPBS for 1 h, then blotted with anti-CLDN12 antibodies at 4 ◦C overnight. After washing, blots were incubated with peroxidase-conjugated goat anti-mouse IgG (1:2000 dilution) at room temperature for 1 h. Immunoblots were developed using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. Biomolecules 2021, 11, 636 4 of 13

2.4. Cell Migration Assays For the cell migration assays of the selected adherent cell lines (A549, LS180, and HeLa), 5.0 × 105 cells/well suspended in 100 µL complete medium (CM) were added in triplicates into the upper chambers of Corning® HTS Transwell® 96 well permeable supports (8.0 µm pore polycarbonate membrane, cat. No CLS3374, Merck KgaA, Darmstadt, Germany) ◦ and incubated at 37 C in a humidified incubator containing 5% CO2 for 24 h. After 24 h of incubation, cell monolayers in the upper chambers were treated with monoclonal anti-CLDN12 antibody (1 µg/mL) for 30 min at 37 ◦C, then washed 3 times with 150 µL serum-free DMEM and incubated for an additional 24 h in 100 µL serum-free DMEM at ◦ 37 C in a humidified atmosphere containing 5% CO2. For the last 24 h of incubation, 100 µL of complete medium was added to the lower chambers. Cells without anti-CLDN12 antibody treatment served as control groups. Cells migrated into the lower chambers were fixed in cold methanol and stained with 0.5% crystal violet for 10 min. All migrated cells were visualized under an Inverso inverted light microscope (Medline Scientific, Chalgrove, Oxfordshire, UK) equipped with a Si-3000 digital camera and software (Medline Scientific, Chalgrove, Oxfordshire, UK). The cells were counted in each transwell of the triplicates and photographed (magnification, ×200). To analyze the migration of Jurkat cells through the tight junctions (containing claudin- 12) of the formed monolayers from the selected adherent cells, we used the same transwell system as described above with small modifications. In order to avoid migration of the adherent cells through the transwell membrane, we used Corning® HTS Transwell® 96 well plates with 3.0 µm pore polycarbonate membrane (cat. No CLS3385, Merck KgaA, Darmstadt, Germany) instead of 8.0 µm. After treatment with monoclonal anti-CLDN12 antibody (blocking the tight junctions) and washing of the cell monolayers formed from A549, LS180, or HeLa cells in the upper chambers, Jurkat cells (1.0 × 106 cells/well) suspended in 150 µL FBS-free medium were added and co-cultured for 24 h at 37 ◦C in a humidified CO2 incubator. A549, LS180, and HeLa cells without anti-CLDN12 antibody treatment served as control groups. Lower chambers were supplemented again with complete medium containing 10% FBS. Migrated Jurkat cells were photographed, counted, and collected for flow cytometric analysis.

2.5. Cell Proliferation Assay An MTT (3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide) assay was used to examine the cell proliferation of the studied cell lines (A549, LS180, and HeLa) after treatment with anti-claudin-12 antibodies or synthetic claudin-12 peptides (p1-p6). Brieﬂy, the cells were seeded (1.0 × 105 cells/well) on 96-well plates (TPP, Trasadingen, ◦ Switzerland) and cultured in complete DMEM for 24 h at 37 C, 5% CO2, and high humidity. Then, we added anti-CLDN12 antibody (1 µg/mL) or synthetic peptides (5 µg/mL, p1-p6) representing the extracellular loops of claudin-12, and incubated the cultures for a further 24 h. For the last 3 h of the incubation period (48 h), 10 µL MTT solution (5 mg/mL) was added to each well. Subsequently, the MTT-containing medium was removed, and 100 µL DMSO was added into each well. The cells were incubated for 15 min at room temperature on a shaker in order to dissolve the accumulated formazan crystals. Absorbance was measured at 540 nm using a Synergy-2 reader (BioTek, Winooski, VT, USA). The results were expressed as a percentage of the untreated control (mean ± SE of triplicates, ** p < 0.01 vs. the control group).

2.6. Flow Cytometric (FACS) Analysis To investigate whether anti-CLDN12 antibodies can induce apoptotic cell death, we used staining with Annexin V-FITC and propidium iodide (PI) followed by ﬂow cytometric analysis. After the treatment period (24 h) with anti-CLDN12 antibodies, A549, LS180, and HeLa cells were washed twice with FACS buffer (DPBS containing 5% fetal calf serum and 0.05% NaN3), harvested by centrifugation at 1500 rpm for 5 min, and resuspended in 500 µL binding buffer. Five microliters of Annexin V-FITC and 5 µL of PI were added Biomolecules 2021, 11, 636 5 of 13

to each sample and the cells were incubated for 15 min at room temperature in darkness. Cells were then washed twice and analyzed using a Cytomics FC 500 Flow Cytometer (Beckman Coulter, Indianapolis, IN, USA). Control Jurkat cells as well as migrated Jurkat cells (after co-culture with the studied adherent cells) were collected and washed with FACS buffer. Then, the cells were stained with FITC-conjugated anti-human CD62L (L-selectin), APC-conjugated anti-human CD11a/CD18 (LFA-1), and PE-conjugated anti-human CD44 antibodies for 30 min at 4 ◦C in darkness. After that, the cells were washed twice with FACS buffer and subjected to ﬂow cytometry using a Cytomics FC500 instrument (Beckman Coulter, Indianapolis, IN, USA). The levels of the studied biomarkers were compared between the control and migrated Jurkat cells.

2.7. Cell Migration Assay with Competitive Inhibition To evaluate the role of claudin-12 protein in the migration of Jurkat cells through the monolayers from adherent cells (A549, LS180, or HeLa), we again performed the same migration assay described before for migration of Jurkat cells, but without treatment of monolayers with anti-CLDN12 antibody. Here, to the co-culture from Jurkat and adherent cells in the upper chambers of the Transwell® (3.0 µm), we added synthetic peptides (p1, p2, p3, p4, p5, p6) representing parts (loop-1 and loop-2) from the extracellular domains of the protein claudin-12 (Figure1). Mimicking the extracellular domains of claudin-12 within the tight junctions of the monolayers formed by A549 and LS180 cells, these peptides compete for binding to the migrated Jurkat cells. If our hypothesis for the involvement of tight junction protein claudin-12 in migration during metastasis was true, the number of migrated Jurkat cells through the tight junctions would be reduced by competitive inhibition. Each peptide was tested in triplicate in a ﬁnal concentration of 5 µg/mL. Co-culture transwells without peptides were used as controls.

2.8. Statistical Analysis All experiments were conducted in triplicate and all data were presented as the mean values ± SE. To compare non-parametric data for statistical signiﬁcance, the Mann-Whitney U-test or Kruskal-Wallis test were applied using the StatView program (SAS Institute). Values of p < 0.05 were considered signiﬁcant (* p < 0.05, ** p < 0.01, *** p < 0.001). All results were compared to those from the controls.

3. Results 3.1. Expression of Claudin-12 In order to select a suitable in vitro model for our study, we tested several human cell lines (Caco-2, LS180, HT-29, A549, HeLa, SK-Hep-1) for expression of claudin-12. Taking into account that this protein expressed predominantly in the tight junctions of epithelia, we chose two claudin-12 expressing cell lines (Figure2a–d) of different origin—A549 (alveolar epithelial cells derived from lung tissue) and LS180 (epithelial cells derived from the colon), and one non-expressing claudin-12 cell line (HeLa) as a negative control (Figure2e–f). In addition to the selected cell lines, Caco-2 and HT-29 cells also expressed claudin-12, although the signal was weaker, but not the cell line SK-Hep-1, which was claudin-12 negative (data not shown). We decided to use the cell line LS180 instead Caco-2 or HT-29 cells because, in addition, LS180 cells produce mucin. To confirm our observations from immunofluorescence, we employed Western blotting analysis for the selected cell lines (A549, LS180, and HeLa). Results demonstrated expression of claudin-12 in both A549 and LS180 cell lines, but not in HeLa cells (Figure3), which corresponded with the data from the immunofluorescence studies. Biomolecules 2021, 11, x FOR PEER REVIEW 6 of 13

ure 2e–f). In addition to the selected cell lines, Caco-2 and HT-29 cells also expressed claudin-12, although the signal was weaker, but not the cell line SK-Hep-1, which was claudin- 12 negative (data not shown). We decided to use the cell line LS180 instead Caco-2 or HT- 29 cells because, in addition, LS180 cells produce mucin.

Biomolecules 2021, 11, x FOR PEER REVIEW 6 of 13

ure 2e–f). In addition to the selected cell lines, Caco-2 and HT-29 cells also expressed clau- Biomolecules 2021, 11, 636 din-12, although the signal was weaker, but not the cell line SK-Hep-1, which was claudin-6 of 13 12 negative (data not shown). We decided to use the cell line LS180 instead Caco-2 or HT- 29 cells because, in addition, LS180 cells produce mucin.

Figure 2. Expression of claudin-12 in A549 (a,b), LS180 (c,d), and HeLa (e,f) cells, cultured for 48 h (a,c,e)—light microscopy; (b,d,f)—fluorescent microscopy). Fixed cells were stained with purified monoclonal anti-CLDN12 antibody and FITC-conjugated secondary anti-mouse IgG. Bar 100 μm.

To confirm our observations from immunofluorescence, we employed Western blot- Figureting analysis2. Expression for of theclaudin-12 selected in A549 cell (a,b lines), LS180 (A549, (c,d), and LS180, HeLa ( e,fand) cells, HeLa). cultured Results for 48 h demonstrated Figure 2. Expression of claudin-12 in A549 (a,b), LS180 (c,d), and HeLa (e,f) cells, cultured for 48 h (a,c,e)—light microscopy; (b,d,f)—fluorescent microscopy). Fixed cells were stained with purified (aexpression,c,e)—light microscopy; of claudin-12 (b,d,f)—ﬂuorescent in both A549 microscopy). and LS Fixed180 cells cell were lines, stained but with not puriﬁed in HeLa cells (Figure monoclonal anti-CLDN12 antibody and FITC-conjugated secondary anti-mouse IgG. Bar 100 μm. monoclonal3), which anti-CLDN12 corresponded antibody with and FITC-conjugatedthe data from secondary the immunofluorescence anti-mouse IgG. Bar 100 µstudies.m. To confirm our observations from immunofluorescence, we employed Western blotting analysis for the selected cell lines (A549, LS180, and HeLa). Results demonstrated expression of claudin-12 in both A549 and LS180 cell lines, but not in HeLa cells (Figure 3), which corresponded with the data from the immunofluorescence studies.

FigureFigure 3. Western3. Western blot analysisblot analysis of claudin-12 of claudin-12 protein demonstrating protein demonstrating expression in A549 expression and LS180 in A549 and LS180 cells.cells. HeLa HeLa cells cells displayed displayed no expression no expression of claudin-12. of claudin-12. Figure 3. Western blot analysis of claudin-12 protein demonstrating expression in A549 and LS180 3.2.cells. Anti-Claudin-12 HeLa cells displayed Antibodies no expression Suppress of the claudin-12. Migration and Proliferation of Claudin-12 Expressing Cancer Cells, Inducing Apoptosis To evaluate whether anti-claudin-12 antibodies can inﬂuence the migration of claudin- 12 expressing cells (A549 and LS180), we used transwell assays. Results showed a sig- Biomolecules 2021, 11, x FOR PEER REVIEW 7 of 13

Biomolecules 2021, 11, 636 3.2. Anti-Claudin-12 Antibodies Suppress the Migration and Proliferation of Claudin-12 7 of 13 Expressing Cancer Cells, Inducing Apoptosis To evaluate whether anti-claudin-12 antibodies can influence the migration of claudin-12 expressing cells (A549 and LS180), we used transwell assays. Results showed a niﬁcant reduction in the number of migrated A549 and LS180 cells after treatment with significant reduction in the number of migrated A549 and LS180 cells after treatment with anti-claudin-12 antibodies compared with non-treated cells (Figure4). anti-claudin-12 antibodies compared with non-treated cells (Figure 4).

Figure 4. Impact of claudin-12 on the migration abilities of cancer cells expressing claudin-12. Figure 4. Impact of claudin-12 on the migration abilities of cancer cells expressing claudin-12. A549, LS180, and HeLa cells were treated with anti-CLDN12 antibody, and the Transwell chamber A549,method LS180, was andused HeLa to evaluate cells were the migration treated with of th anti-CLDN12e treated and antibody, non-treated and cells. the TranswellCells were chamber stained methodwith 0.5% was crystal used toviolet evaluate (a,b,d the,e,g migration,h). Results of from the treatedthe comparative and non-treated analysis cells. (c,f,i) Cells are presented were stained as withmean 0.5% ± standard crystal violeterror of (a ,theb,d ,mean.e,g,h). *** Results indicates from p the< 0.001. comparative Bar 100 μ analysism. (c,f,i) are presented as mean ± standard error of the mean. *** indicates p < 0.001. Bar 100 µm. The mean numbers of migrated A549 cells with and without antibody treatment were 23 ±The 4 and mean 193 numbers ± 17 cells, of respectively migrated A549 (Figure cells with4a–c). and For without the LS180 antibody cell line, treatment these values were 23were± 4 6 and ± 3 193and± 28317 cells,± 8 cells, respectively respectively (Figure (Figure4a–c). 4d–f). For Our the LS180data revealed cell line, no these significant values weredifference 6 ± 3 andin migration 283 ± 8 cells,ability respectively between the (Figure anti-claudin-124d–f). Our antibody data revealed treatedno (133 signiﬁcant ± 10) and differencenon-treated in migration(165 ± 7) HeLa ability cells between (Figure the 4d–f). anti-claudin-12 These analyses antibody suggest treateded that (133 claudin-12± 10) and non-treatedin the tight(165 junctions± 7) HeLa is involved cells (Figure in the 4migrationd–f). These ofanalyses cells expressing suggested this that protein. claudin-12 in the tightIn addition, junctions to is involvedexamine the in the effect migration of anti-c oflaudin-12 cells expressing antibodies this on protein. cell viability (respectively,In addition, cell proliferation), to examine the we effect performed of anti-claudin-12 MTT assays antibodies using the on same cell viabilitystrategy (respec-of anti- tively,body celltreatment. proliferation), As shown we in performed Figure 5, antibody MTT assays treatment using re thesulted same in strategy a significant of antibody reduc- treatment.tion in proliferation As shown inof Figureclaudin-125, antibody expressing treatment A549 and resulted LS180 in cells a signiﬁcant compared reduction to the con- in proliferationtrols, and a ofslight claudin-12 increase expressing in proliferation A549 andof HeLa LS180 cells. cells After compared 48 h, tothe the cell controls, viability and of aA549 slight cells increase decreased in proliferation to 35.47%, of and HeLa of cells.LS180 After cells, 48to h,26.42% the cell (Figure viability 5). ofMTT A549 assays cells decreaseddemonstrated to 35.47%, that anti-claudin-12 and of LS180 cells,antibodies to 26.42% suppress (Figure cell5 proliferation). MTT assays as demonstrated well. that anti-claudin-12 antibodies suppress cell proliferation as well. Biomolecules 2021, 11, 636 8 of 13 BiomoleculesBiomolecules 2021 2021, ,11 11, ,x x FOR FOR PEER PEER REVIEW REVIEW 88 ofof 1313

FigureFigure 5. 5. Impact Impact of of claudin-12 claudin-12 on on the the proliferation proliferation of of cancer cancer cells cells expressi expressingng claudin-12. claudin-12. A549, A549, Figure 5. Impact of claudin-12 on the proliferation of cancer cells expressing claudin-12. A549, LS180, LS180,LS180, and and HeLa HeLa cells cells were were trea treatedted with with anti-CLDN12 anti-CLDN12 antibody, antibody, and and the the MTT MTT assay assay was was used used to to and HeLa cells were treated with anti-CLDN12 antibody, and the MTT assay was used to examine the examineexamine the the cell cell proliferation proliferation of of the the treated treated and and non-treated non-treated cells. cells. Results Results are are presented presented as as a a per- per- cellcentagecentage proliferation of of viable viable of cells cells the compared treatedcompared and to to non-treated the the control control cells.(n (non-treatedon-treated Results arecells)cells) presented calculated calculated as from afrom percentage triplicates. triplicates. of viable** ** cellsindicatesindicates compared p p < < 0.01. 0.01. to the control (non-treated cells) calculated from triplicates. ** indicates p < 0.01. To evaluate whether the reduced cell viability is due to apoptosis or necrosis, we ToTo evaluateevaluate whetherwhether thethe reducedreduced cellcell viabiviabilitylity isis duedue toto apoptosisapoptosis oror necrosis,necrosis, wewe conducted ﬂow cytometry. Double staining with Annexin-V FITC and propidium iodide conductedconducted flowflow cytometry.cytometry. DoubleDouble stainingstaining withwith Annexin-VAnnexin-V FITCFITC andand propidiumpropidium iodideiodide clearly indicated that anti-CLDN12 antibodies induce apoptosis in A549 (8.85%) and LS180 clearlyclearly indicatedindicated thatthat anti-CLDN12anti-CLDN12 antibodiesantibodies induceinduce apoptosisapoptosis inin A549A549 (8.85%)(8.85%) andand cells (25.3%) (Figure6), probably via blockade of cell division after binding to claudin-12. LS180LS180 cells cells (25.3%) (25.3%) (Figure (Figure 6), 6), probably probably via via blockade blockade of of cell cell division division after after binding binding to to clau- clau- HeLa cells were not affected (Figure6). These data suggest that the inhibition of cell din-12.din-12. HeLaHeLa cellscells werewere notnot affectedaffected (Figure(Figure 6)6).. TheseThese datadata suggestsuggest thatthat thethe inhibitioninhibition ofof proliferation and migration of claudin-12 expressing cells is due to apoptotic cell death cellcell proliferation proliferation and and migration migration of of claudin-12 claudin-12 expressing expressing cells cells is is due due to to apoptotic apoptotic cell cell death death caused by anti-CLDN12 antibodies. causedcaused byby anti-CLDN12anti-CLDN12 antibodies.antibodies.

FigureFigureFigure 6.6. 6. Apoptotic Apoptotic effects effects of of anti-CLDN12 anti-CLDN12anti-CLDN12 antibodies antibodies on on A549, A549, A549, LS180, LS180, LS180, and and and He He HeLaLaLa cells. cells. cells. The The The cells cells cells were treated with anti-CLDN12 antibodies (1 μg/mL) for 24 h and then stained with annexin V- werewere treated treated with with anti-CLDN12 anti-CLDN12 antibodies antibodies (1 (1µg/mL) μg/mL) for for 24 24 h andh and then then stained stained with with annexin annexin V-FITC V- FITCFITC (horizontal (horizontal arrow) arrow) and and prop propidiumidium iodide iodide (PI, (PI, vertical vertical arrow). arrow). The The percentages percentages of of apoptotic apoptotic (horizontal arrow) and propidium iodide (PI, vertical arrow). The percentages of apoptotic cells are cellscells are are shown shown on on the the histograms histograms (second (second row). row). shown on the histograms (second row). 3.3.3.3. Claudin-12Claudin-12 IsIs InvolvedInvolved inin thethe MigrationMigration ofof JurkatJurkat CellsCells ThroughThrough thethe TightTight JunctionsJunctions ToTo determinedetermine thethe involvementinvolvement ofof claudin-12claudin-12 inin thethe processprocess ofof metastasis,metastasis, wewe nextnext per-per- formedformed assaysassays similarsimilar toto thethe previousprevious trantranswellswell assays,assays, butbut usingusing membranesmembranes withwith 3.03.0 μμmm Biomolecules 2021, 11, 636 9 of 13

3.3. Claudin-12 Is Involved in the Migration of Jurkat Cells through the Tight Junctions

Biomolecules 2021, 11, x FOR PEER REVIEWTo determine the involvement of claudin-12 in the process of metastasis, we9 nextof 13 performed assays similar to the previous transwell assays, but using membranes with 3.0 µm pore size and a co-culture of adherent cells (A549, LS180, or HeLa) and Jurkat cells (Figure7). The results revealed that Jurkat cells were able to migrate mostly through pore size and a co-culture of adherent cells (A549, LS180, or HeLa) and Jurkat cells (Figure 7). the tight junctions of the claudin-12 expressing cells (A549 and LS180) that were not pre- The results revealed that Jurkat cells were able to migrate mostly through the tight junctions treated with anti-claudin-12 antibodies (Figure7). The mean numbers of Jurkat cells that of the claudin-12 expressing cells (A549 and LS180) that were not pre-treated with anti-clau- migrated through A549 and LS180 cells were 310 ± 30 (Figure7c) and 254 ± 21, respectively din-12 antibodies (Figure 7). The mean numbers of Jurkat cells that migrated through A549 (Figure7f). and LS180 cells were 310 ± 30 (Figure 7c) and 254 ± 21, respectively (Figure 7f).

Figure 7. Impact of claudin-12 on the migration abilities of Jurkat cells. Monolayers from A549, Figure 7. Impact of claudin-12 on the migration abilities of Jurkat cells. Monolayers from A549, LS180, and HeLa cells were treated with anti-CLDN12 antibody and co-cultured with Jurkat cells. LS180,The transwell and HeLa chamber cells were method treated was with used anti-CLDN12 to evaluate the antibody migration and of co-cultured Jurkat cells withthrough Jurkat the cells. Themonolayers. transwell Images chamber were method taken wasfrom usedthe lower to evaluate chambers the where migration the migrated of Jurkat cells cells should through be the monolayers.(a,b,d,e,g,h). ImagesResults werefrom the taken comparative from the analysis lower chambers (c,f,i) are wherepresented the as migrated mean ± standard cells should error be (a,ofb, dthe,e, gmean.,h). Results *** indicates from the p < comparative0.001. Bar 100 analysis μm. (c,f,i) are presented as mean ± standard error of the mean. *** indicates p < 0.001. Bar 100 µm. We found no differences in the co-culture system HeLa-Jurkat, where migrated JurkatWe foundcells were no differences not detected in the(Figure co-culture 7g–i). systemThese results HeLa-Jurkat, confirmed where that migrated claudin-12 Jurkat is cellsinvolved were not in cell detected migration (Figure during7g–i). metastasis. These results conﬁrmed that claudin-12 is involved in cell migration during metastasis. 3.4. Migrating Jurkat Cells Express Lymphocyte Function-Associated Antigen-1 (LFA-1 3.4.Integrin) Migrating and JurkatL-Selectin Cells (CD62L) Express Lymphocyte Function-Associated Antigen-1 (LFA-1 Integrin) and L-Selectin (CD62L) We also performed flow cytometric analysis of the Jurkat cells for expression of LFA- 1 andWe CD62L also performed before co-culturing ﬂow cytometric and after analysis the migration of the Jurkat assay. cells As for can expression be seen in of Figure LFA-1 and8, Jurkat CD62L cells before constitutively co-culturing express and afterthe integrin the migration LFA-1 (before assay. Asand can after be migration), seen in Figure while8, the expression of L-selectin (CD62L) was induced after co-culturing with A549 or LS180 cells. These results suggest that for migration through the tight junctions, Jurkat cells probably use LFA-1 or CD62L, or both molecules. Jurkat cells did not express CD44 glycoprotein (data not shown). Biomolecules 2021, 11, 636 10 of 13

Biomolecules 2021, 11, x FOR PEER REVIEW 10 of 13 Jurkat cells constitutively express the integrin LFA-1 (before and after migration), while the expression of L-selectin (CD62L) was induced after co-culturing with A549 or LS180 cells. These results suggest that for migration through the tight junctions, Jurkat cells probably Biomolecules 2021, 11, x FOR PEER REVIEW 10 of 13 use LFA-1 or CD62L, or both molecules. Jurkat cells did not express CD44 glycoprotein (data not shown).

Figure 8. Flow cytometric analysis of migrated Jurkat cells (shaded histograms) and control Jurkat

cells (open histograms) for expression of LFA-1 and L-selectin. Cells were stained with anti-hu- FiguremanFigure CD11a/CD18 8. 8.Flow Flow cytometric cytometric APC-conjugated analysis analysis of of migrated migrated (for LFA-1) Jurkat Jurkat an cells dcells anti-human (shaded (shaded histograms) histograms) CD62L FITC-conjugated and and control control Jurkat Jurkat (for L- cells (open histograms) for expression of LFA-1 and L-selectin. Cells were stained with anti-hu- cellsselectin) (open antibodies. histograms) The for expressiondata are fr ofom LFA-1 one andrepresentative L-selectin. Cells experiment. were stained with anti-human man CD11a/CD18 APC-conjugated (for LFA-1) and anti-human CD62L FITC-conjugated (for L- CD11a/CD18 APC-conjugated (for LFA-1) and anti-human CD62L FITC-conjugated (for L-selectin) selectin) antibodies. The data are from one representative experiment. antibodies.3.5. Claudin-12 The data Peptides are from Can one representativeBlock the Migratio experiment.n of Jurkat Cells Through the Tight Junctions 3.5. Claudin-12 Peptides Can Block the Migration of Jurkat Cells Through the Tight Junctions 3.5. Claudin-12To examine Peptides the Canpotential Block the of Migrationclaudin-12 of Jurkat peptides Cells Through(derived the from Tight the Junctions extracellular do- To examine the potential of claudin-12 peptides (derived from the extracellular do- mainsTo of examine the claudin-12 the potential protein) of claudin-12 to reduce peptides the migration (derived ability from theof Jurkat extracellular cells, we do- repeated mainsthemains transwell of of the the claudin-12 claudin-12 experiments protein) protein) with to to reduceco-culture reduce the the migration systems,migration ability but ability instead of of Jurkat Jurkat of cells,anti-claudin-12 cells, we we repeated repeated antibod- theies,the transwell we transwell used experiments synthetic experiments claudin-12 with with co-culture co-culture peptides systems, systems, (Figure but but instead 9). instead Our of resultsof anti-claudin-12 anti-claudin-12 indicated antibod- antibod-that two of the ies,usedies, we we peptides used used synthetic synthetic (p5 and claudin-12 claudin-12 p6) completely peptidespeptides inhibited (Figure9 9).). the Our migration results results indicated indicated of Jurkat that that cells two two ofthrough of the the theA549used used (Figurepeptides peptides 9a) (p5 (p5 and and and LS180p6) p6) completely completely cells (Figure inhibited inhibited 9b). theAlthough the migration migration the of other ofJurkat Jurkat claudin-12 cells cells through through peptides the (p1, A549 (Figure 9a) and LS180 cells (Figure 9b). Although the other claudin-12 peptides (p1, thep2, A549 p3, and (Figure p4)9a) slightly and LS180 reduced cells (Figure the migration9b). Although of Jurkat the other cells claudin-12 compared peptides to the control (p1,p2, p2, p3, p3, and and p4) p4) slightly slightly reduced reduced the the migration migration of Jurkat cellscells comparedcompared to to the the control control (black(black bars,bars, withoutwithout peptides), peptides), this this inhibition inhibition was was not significantnot significant (Figure (Figure 9). Results 9). Results sug- sug- (blackgest that bars, Jurkat without cells peptides), migrate this through inhibition the was ti notght significant junctions (Figure by binding9). Results to suggestclaudin-12 and thatgest Jurkat that Jurkat cells migrate cells migrate through through the tight the junctions tight junctions by binding by binding to claudin-12 to claudin-12 and more and more specifically, to the first extracellular loop (Figure 1), because the peptides p5 and p6 specifically,more specifically, to the first to the extracellular first extracellular loop (Figure loop (Figure1), because 1), because the peptides the peptides p5 and p5 p6 and are p6 partsareare parts parts of this of domain. this domain. domain. The peptideThe The peptide peptide p1 (red p1 p1(red bars) (red bars) that bars) that represented thatrepresented represented the secondthe second the extracellular second extracellu- extracellu- looplarlar loop didloop not did inhibit notnot inhibit theinhibit migration the the mi migration ofgration Jurkat of cells. Jurkatof Jurkat As cells. shown cells. As in shown FigureAs shown 9ind, Figure the in used Figure 9d, synthetic the 9d, used the used peptidessyntheticsynthetic did peptides not have did did a significantnot not have have a impact significanta significant on the impact claudin-12 impact on the on expressing claudin-12the claudin-12 cells expressing (A549 expressing and cells cells LS180).(A549(A549 and Decreasedand LS180). cell DecreasedDecreased viability (65%cell cell viability from viability the (65% control) (65% from wasfrom the measured control)the control) was after measured was treatment measured after of after HeLatreatmenttreatment cells with of HeLaHeLa p5. At cells cells this with point,with p5. wep5. At doAt this not this point, have point, anwe explanation dowe not do havenot as havean it wasexplanation an irrelevant explanation as to itthe was as it was tasksirrelevantirrelevant of this to study. thethe tasks tasks of of this this study. study.

Figure 9. Cont.

Biomolecules 2021, 11, x FOR PEER REVIEW 10 of 13

Figure 8. Flow cytometric analysis of migrated Jurkat cells (shaded histograms) and control Jurkat cells (open histograms) for expression of LFA-1 and L-selectin. Cells were stained with anti-human CD11a/CD18 APC-conjugated (for LFA-1) and anti-human CD62L FITC-conjugated (for L- selectin) antibodies. The data are from one representative experiment.

3.5. Claudin-12 Peptides Can Block the Migration of Jurkat Cells Through the Tight Junctions To examine the potential of claudin-12 peptides (derived from the extracellular domains of the claudin-12 protein) to reduce the migration ability of Jurkat cells, we repeated the transwell experiments with co-culture systems, but instead of anti-claudin-12 antibodies, we used synthetic claudin-12 peptides (Figure 9). Our results indicated that two of the used peptides (p5 and p6) completely inhibited the migration of Jurkat cells through the A549 (Figure 9a) and LS180 cells (Figure 9b). Although the other claudin-12 peptides (p1, p2, p3, and p4) slightly reduced the migration of Jurkat cells compared to the control (black bars, without peptides), this inhibition was not significant (Figure 9). Results suggest that Jurkat cells migrate through the tight junctions by binding to claudin-12 and more specifically, to the first extracellular loop (Figure 1), because the peptides p5 and p6 are parts of this domain. The peptide p1 (red bars) that represented the second extracellular loop did not inhibit the migration of Jurkat cells. As shown in Figure 9d, the used synthetic peptides did not have a significant impact on the claudin-12 expressing cells (A549 and LS180). Decreased cell viability (65% from the control) was measured after treatment of HeLa cells with p5. At this point, we do not have an explanation as it was irrelevant to the tasks of this study.

Biomolecules 2021, 11, 636 11 of 13

Figure 9. Migration capacity of Jurkat cells after incubation with synthetic claudin-12 peptides (a–c) and effects of the peptides on A549, LS180, and HeLa cells (d). Monolayers from A549 (a), LS180 (b), and HeLa cells (c) were co-cultured for 24 h with Jurkat cells in the presence of 5 µg/mL synthetic claudin-12 peptides. Six synthetic claudin-12 peptides (p1-p6) representing the extracellular domains of the claudin-12 protein were tested as competitors of the claudin-12 in the tight junctions. The transwell chamber method was used to evaluate the migration of Jurkat cells through the monolayers. Results are presented as mean ± standard error of the mean. *** indicates p < 0.001. (d) A549, LS180, and HeLa cells were treated with 5 µg/mL synthetic claudin-12 peptides (p1–p6) for 24 h, and the MTT assay was used to examine the cell proliferation of the treated and non-treated cells. Results are shown as a percentage of viable cells compared to the control (non-treated cells) calculated from triplicates.

4. Discussion Claudin proteins are integral components of the tight junctions maintaining cell polarity, paracellular permeability, cell proliferation, transformation, and metastasis [21]. It has been shown that during metastasis, expression of certain claudins could increase or decrease in a tissue-specific fashion [17,22–30]. The expression and functions of these proteins are regulated by different mechanisms including disintegration of the cell-cell contacts, cytokines, hormones, or other signaling pathways. In our study, we focused on the non-canonical claudin-12 (lacking a PDZ binding domain) and its significance for cell migration during metastasis. We found that epithelial-derived cancer cell lines from the colon (LS180, Caco-2, HT- 29) and the lung (A549) express claudin-12, while liver endothelial SK-Hep-1 and cervix epithelial HeLa cancer cells were claudin-12 negative. This is in agreement with results of other studies that showed expression of claudin-12 in the epithelia and endothelia of the gastrointestinal tract [1] or bronchial epithelial cells [17]. Lack of expression of claudin proteins in HeLa cells (claudin-null cell line) was also previously reported [31,32]. This comparative analysis showed that the cell lines (A549, LS180, HeLa) used as models in this study were correctly selected. Pretreatment of claudin-12 expressing cells (A549, LS180) with anti-claudin-12 antibodies significantly reduced both migration and proliferation of these cells and induced apoptosis, probably blocking the cytokinesis of cell division after binding to the extracellular domain of claudin-12. Even more, such pretreatment blocked the migration of Jurkat cells through the tight junctions of the formed epithelial cell monolayers during the co-culture. These observations are novel and demonstrate the potential capabilities of anti-claudin-12 antibodies to inhibit the metastatic process in claudin-12 expressing tissues. For example, it has recently been reported that claudin-12 is involved in the epithelial-mesenchymal transition and migration of human bronchial epithelial BEAS-2B cells [17]. In addition, the same researchers found upregulated expression of claudin-12 in lung squamous cell carcinoma (SqCC) tissues, suggesting the CLDN12 gene as a proto- oncogene in SqCC [17]. Similarly, Tian et al. concluded that cytoplasmic overexpression of claudin-12 promotes the proliferation and migration ability of osteosarcoma cells [14]. Taking into account the tissue-specific expression of the claudin proteins, Jiang et al. [21] noted that they could be used as prognostic and diagnostic biomarkers, e.g., claudin-1 for Biomolecules 2021, 11, 636 12 of 13

colon cancers, claudin-3 for ovarian cancers, claudin-10 for hepatocellular carcinomas, etc. Thus, claudin-12 may be used as a biomarker for tumor progression and metastasis in the gastrointestinal tract, lung SqCC, and osteosarcomas. On the other hand, claudin proteins were identified as an attractive target for antitumor therapy [21]. Therefore, blocking expression of claudin-12 with anti-claudin-12 antibodies should have a beneficial effect, inhibiting tumor progression and metastasis in tissues expressing claudin-12. Our results showed that short peptides from the first extracellular loop (Figure1) of the protein claudin-12 are also able to reduce the migration of Jurkat cells through the tight junctions, suggesting that competitive inhibition mechanisms could be useful in the therapeutic approach applied to cancer metastasis. We found that migrating Jurkat cells express both integrin LFA-1 and L-selectin (CD62L). It is well known that LFA-1 and L-selectin play a major role in the adhesion of circulating leukocytes to the endothelial cells regulating T cell activation and migration through the endothelium [33–35]. Based on our observation that anti-claudin-12 antibodies reduced the number of migrated Jurkat cells, we hypothesized that claudin-12 is a ligand for LFA-1 and/or L-selectin. However, additional studies are needed to prove whether claudin-12 binds to LFA-1 and L-selectin, leading to the disruption of tight junctions and migration of T cells.

5. Conclusions We have shown that A549 and LS180 cells express claudin-12, and that anti-claudin-12 antibodies inhibit cell migration and proliferation and induce apoptosis of claudin-12 expressing cells. Furthermore, synthetic peptides representing the first extracellular loop of claudin-12 reduce the migration of Jurkat cells. Migrating Jurkat cells express LFA-1 and L-selectin. Our findings identified the essential role of claudin-12 in the migration of cancer cells through claudin-12 expressing tissues in the process of metastasis.

Author Contributions: Conceptualization, B.D.; methodology, B.D. and T.B.; formal analysis, D.K., D.M. and T.B.; investigation, D.K., D.M. and T.B.; supervision, T.B. and B.D.; writing—original draft preparation, B.D. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the operational program “Science and education for smart growth” 2014–2020, grant number BG05M2OP001-1.002-0005-C01, Personalized Innovative Medicine Competence Center (PERIMED). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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