Supplement Results, Figures and Tables
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1 Supplement Results, Figures and Tables Results PET imaging The SUV was significantly higher in tumours of the parental HSC-3 cell line than tumours of the EV and the MMP-8 overexpressing cell lines (Figure S4B upper panel), suggesting that the cloning procedure somehow affected the metabolism of the cells. Tongue tumours normally metastasize to lymph nodes in the neck, which overlap the sublingual and parotid salivary glands in mice. Salivary glands give background signal in 18 F-FDG PET-scans due to their metabolic activity. To identify lymph node metastases, the salivary gland area was delineated and the 18 F-FDG uptake in left and right salivary gland areas were compared (Figure S4A lower panel). In mice with tumours of the HSC-3 parental and control cell lines, there was more uptake in the left side compared to the right side, although the differences were not statistically significant (Figure S4B, lower panel). In mice injected with the MMP-8+ HSC-3 cells, the 18 F- FDG uptake was similar on the left and right side. However, the differences were not statistically significant. Figure S4C shows representative time-activity curves of 18 F-FDG uptake in liver, tumour and in the left and right salivary glands in an OSCC xenograft mouse based on a 60 min dynamic PET scan. This illustrates that the 20 min static scans, starting at about 40 min after 18 F-FDG injection, were within a stationary phase of the 18 F-FDG uptake. Figures Figure S1. The overexpressed prommp-8 level is higher in conditioned media of HSC-3 clone #1 compared to clone #2, and pro-mmp-8 was activated after treatment with APMA in MMP-8+ cells. Clone #1 of MMP-8+ HSC-3 cells showed higher expression of MMP-8 compared to the clone #2 cells in Western blot analysis (A). APMA activation led to reduction of prommp-8 (~70kDA) and increase of mature MMP-8 (~58kDa) as shown by Western blot (B). 1
2 Figure S2. Apoptosis, EMT markers and proliferation in control and MMP-8+ OTSCC cells. Apoptosis was studied by TUNEL technology from cells seeded into wells of 96-well plates as described in the methods (A). The levels of E-cadherin, Slug, N-cadherin and vimentin were not changed in cell extracts (40 µg protein) studied by Western blot from control and MMP-8+ HSC-3, SCC-25 and SCC-15 cells cultured in monolayers. Representative results are shown (B). Levels of immunohistochemically detected vimentin was slightly reduced in MMP- 8+ HSC-3 cells in organotypic 3D tissue cultures. In control cells, vimentin was stained in invasive cells (arrow), but the staining disappeared from invasive MMP-8+ HSC-3 cells; the amount of E-cadherin was not changed (C). Proliferation of HSC-3 cells was studied by BrdU kit. Two to four independent experiments (with different cell passages) were studied at each time point (D). Six samples of each cell group were analysed in the TUNEL assay. Western blots were performed using cell extracts from four different individual experiments. The myoma experiment was performed once with triplicate myoma disks per culture condition. 2
3 Figure S3. MMP-8 overexpression reduced the migration of HSC-3 cells and altered the response to TGF-β1 in clone #2. 10 ng/ml of recombinant human TGF-β1 was added to the serum-free medium in the upper Transwell chamber and the amount of migrated MMP-8+ HSC-3 and control cells was measured as absorbance at 650 nm. * p < 0.05, ** p < 0.01, *** p <
4 Figure S4. Evaluation of 18 F-FDG uptake and temporal pattern of 18 F-FDG uptake in OSCC xenograft mice (example graph). Coronal PET sections co-registered with a maximum intensity projection (MIP) CT image of an OSCC xenograft mouse showing average tumour and salivary gland (Sg) 18 F-FDG uptake min post injection (A). Tumour and salivary gland 18 F-FDG uptake (SUVmax) relative to liver uptake (SUVmean) in the different OTSCC cell groups. Lower panel, left salivary glands (filled bars) and right salivary glands (shaded bars). Results are mean ± SEM. *p<0.05, **p<0.01. Sg, salivary glands (B). An example graph shows representative time-activity curves of 18 F-FDG uptake in liver (grey symbols), tumour (red symbols), left (blue closed symbols) and right (blue open symbols) salivary glands (sg) in an OTSCC xenograft mouse (MMP-8+ HSC-3 group) during a 60 min PET acquisition (C). 4
5 Figure S5. MMP-8 immunohistochemical staining of HSC-3 tumours in mouse tongues. Tumours of the HSC-3 parental cell line (A-B) and the control cell line (C-D) did not show cytoplasmic MMP-8 staining of the cancer cells. In the tumours of MMP-8 overexpressing cells, MMP-8 staining was mainly seen in the cells located in the centre of tumour islands. In the tumour-stroma interface, the cancer cells showed weak or absent cytoplasmic MMP-8 staining. Some of the mouse inflammatory cells were positive for MMP-8 (E-F). Some nuclear staining could be seen in the cancer cells of all clones. 5
6 Figure S6. H/E staining of the primary tumours and lymph node metastasis in mice with MMP-8+ and control HSC-3 clones. Representative primary tumours and lymph node metastases in mice with MMP-8+ and control cells (A). The lymph nodes were analysed after 13 days to count the number of mice with and without metastases (B). The areas of primary tumours and metastases were measured (C). 6
7 Tables Table S1. Patients clinical data (OTSCC samples used in the VEGF-C and MMP-8 analyses). Baseline patient characteristic Patient clinical data n % Total Age at diagnosis <55 yrs yrs >70 yrs Sex Male Female Tumour grade Tumour stage Neck lymph nodes Negative Positive Recurrence No Yes Adjuvant therapy No Radiotherapy Missing
8 Table S2. Differentially expressed genes in MMP-8 overexpressing stationary and migrating cells. Top 20 genes upregulated in MMP-8 overexpressing cells compared to controls Stationary cells (43 a ) Migrating cells (40 a ) GJB6 b, CXCL14, FOS b, KRT13, PHC1, GJB2, GJB6 b, TUSC1 b, TENM2 b, KLK10, KLK13 b, S100A8, EGR1, MEST b, KLK13 b, TUSC1 b, S100A8, TENM2 b, MAGED1 b, EGR1, MEST b, IL17D b, GJB2 b, SCEL b, BLNK, ARMCX1 b, MAGED1 b, KLK10, ID1, GJA1 b, BCHE b, GAPDHS b, NRG1, GAPDHS b, NRG1, IL1RN, BCHE b, MCAM VGLL1, IL27RA b, ARMCX1 b, FLRT3 b Top 20 genes downregulated in MMP-8 overexpressing cells compared to controls Stationary cells (40 a ) Migrating cells (39 a ) MMP1 b, RBM15B b, MALAT1 b, LIMCH1 b, L1CAM b, MMP1 b, L1CAM b, NNMT b, MAP2 b, AGPAT9, CFB, NNMT b, CFB, LYN b, C3 b, KLRC1/2, MAP2 b, SRPX b, LYN b, POLI b, C3 b, KLRC1/2, PRTFDC1 b, ALS2CL b, TNFAIP3, FAM20C b, POLI b, UGT1A1/3/4/5/6/7/8/9/10, FLNC, UGT1A1/3/4/5/6/7/8/9/10, MUC1, GRIP2 b, BIRC3, C18orf54, NRG1, SNHG12 FADS3, C18orf54 b, CROCC, POMZP3/ZP3 Changed genes only in migrating or stationary cells, top 15 =upregulated, top 15 = downregulated MMP-8 overexpressing cells vs. control cells Change only in stationary cell (42 a ): : CXCL14, FOS, KRT13, PHC1, BLNK, MCAM, LTBP1, NIN, TFCP2L1, HES1, FAM213A, CHCHD10, C6orf62, SSBP3, BCL11B : RBM15B, MALAT1, LIMCH1, SRPX, TNFAIP3, FAM20C, BIRC3, SNHG12, ABLIM3, SLC7A11,SLC39A8, PPAP2B, INSIG1,TWIST1, SLC47A2 Change only in migrating cells (37 a ): : IL17D, SCEL, GAPDHS, TBX1, DOCK1, KREMEN1, MMP9, BAK1, ELAVL2, PTPLB, FAM89A, FAM101B, ASNS, MRPL43, TFPI2 : AGPAT9, PRTFDC1, FLNC, MUC1, ALS2CL, FADS3, POMZP3/ZP3, CROCC, SERPINA1, TMEM123, LOC284591, IL7R, CSRP2, NEXN, AKR1B10 a Total number of changed genes (approx.) with fold change value
9 Table S3. Differentially expressed genes in MMP-8 overexpressing cells involved in cancer-related events. The changed genes (MMP-8+ vs. control cells) that are involved in cancer-related cell processes Function Overexpressed Underexpressed Stationary cells Migrating cells Stationary cells Migrating cells Actin cytoskeletal FLRT3, LAMA2 LIMCH1 organisation Cell adhesion HES1, FLRT3 BCAM L1CAM, OLR1 L1CAM Cell motion ID1 TBX1, GAPDHS, ID1 IL8, L1CAM, VEGFC, L1CAM PPAP2B, TWIST1, NDE1 Transcriptional regulation EGR1, HES1, ELAVL2, TBX1, SOD2, TWIST1, YAP1, SOD2, VGLL1, MAGED1, FOS, ID1, EGR1, MAGED1, LRF1, YAP1, TRIM22 BCL11B, TFCP2L1 ID1, VGLL ABLIM3, Regulation of NRG1 NRG1, PAPPA2 cell growth Inflammatory / S100A8 S100A8, IL1RN, CFB, LYN, IL8, CFB, SERPINA1, C3, immune resp. IL1RN IL27RA PTX3, OLR1, C3 IL7R, LYN, TRIM22 Proteolysis KLK10, KLK5 KLK10, KLK13, ECE1, ISG15, SENP7 CFB, MMP1, C3 KLK5, MMP9 OLR1, CFB, PAPPA2 TNFAIP3, MMP1, C3 9
10 Table S4. Clinical correlations with tumour MMP-8 and VEGF-C status. MMP-8 expression VEGF-C expression Weak, n (%) Strong, n (%) p-value Weak, n (%) Strong, n (%) p value Age at diagnosis <55 yrs 9 (45.0) 11 (55.0) 11 (55.0) 9 (45.0) yrs 11 (78.6) 3 (21.4) 7 (50.0) 7 (50.0) >70 yrs 14 (60.9) 9 (39.1) (43.5) 13 (56.5) Sex Male 13 (52.0) 12 (48.0) 15 (60.0) 10 (40.0) Female 21 (65.6) 11 (34.4) (40.6) 19 (59.4) Tumour grade 1 12 (54.5) 10 (45.5) 12 (54.5) 10 (45.5) 2 18 (62.1) 11 (37.9) 13 (44.8) 16 (55.2) 3 4 (66.7) 2 (33.3) (50.0) 3 (50.0) Tumour stage (61.3) 12 (38.7) 18 (58.1) 13 (41.9) (57.7) 11 (42.3) (38.5) 16 (61.5) Neck lymph nodes Negative 23 (53.5) 20 (46.5) 25 (58.1) 18 (41.9) Positive 11 (78.6) 3 (21.4) (21.4) 11 (78.6) Recurrence No 21 (55.3) 17 (44.7) 22 (57.9) 16 (42.1) Yes 13 (68.4) 6 (31.6) (31.6) 13 (68.4) Adjuvant therapy No 21 (55.3) 17 (44.7) 21 (55.3) 17 (44.7) Radiotherapy 10 (62.5) 6 (37.5) (37.5) 10 (62.5)
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