Fig 2: HDFs with Opposite Modulation of FGF versus TGF-ß Signaling Stimulate In Vivo SCC Cell Proliferation, Eliciting Different Tumor Microenvironments(A–F) Immunofluorescence analysis of the same nodules as in Figures 6E–6J for a-SMA (DNFGFR1 in A and DNTGFB2 in B), Periostin (POSTN) (DNFGFR1 in C and DNTGFB2 in D), or MMP1 (DNFGFR1 in E and DNTGFB2 in F) with concomitant staining for E-cadherin or pan-keratin for SCC cell visualization as indicated. Shown are representative images (left panels) and quantifications (right panels) of a-SMA-, Periostin-, or MMP1-positive areas. Scale bar, 100 µm. n (nodules per conditions, 4 fields per nodule) = 4, mean ± SEM, two-tailed paired t test, *p < 0.05, **p < 0.005, ***p < 0.001.(G and H) Double immunofluorescence analysis of the same nodules as in the previous panels for pan-keratin (red) and vimentin (green). Shown are representative images (left panels) and quantification (right panels) of the fraction of pan-keratin and vimentin double-positive cells with DNFGFR1-expressing (G) and DNTGFBR2-expressing (H) HDFs versus controls. Scale bar, 100 µm. n (nodules per conditions, 4 fields per nodule) = 5, mean ± SEM, two-tailed paired t test, *p < 0.05.(I–L) Immunofluorescence analysis of the same nodules as in the previous panels for F4/80 (DNFGFR1 in I and DNTGFBR2 in J) or CD206 (DNFGFR1 in K and DNTGFBR2 in L). Shown are representative images (left panels) and quantifications (right panels) of F4/80- and CD206-positive areas. Scale bar, 100 µm. n (nodules per conditions, 4 fields per nodule) = 4, mean ± SEM, two-tailed paired t test, *p < 0.05, **p < 0.005.(M and N) Fluorescence-guided LCM of HDFs labeled with anti-PDGFRa-fluorescein isothiocyanate (FITC)-conjugated antibodies of consecutive sections of the same nodules as in the previous panels followed by qRT-PCR analysis of indicated genes. (M) Representative images of a PDGFRa-FITC-stained section with propidium iodide for nuclear visualization before capturing and of fluorescence and phase contrast images of captured cells on an LCM cap. (N) Heatmap of qRT-PCR analysis of ETV1 and indicated CAF effector genes in the captured fibroblasts, with values expressed as Log10 ratios of DNFGFR1- or DNTGFBR2-expressing HDFs versus controls. n (nodules per conditions) = 3. Scale bars, 150 µm (M, left and center) and 750 µm (M, right).(O) Fluorescence-guided LCM of SCC13 cells labeled with anti-pan-keratin-Alexa 488-conjugated antibodies of consecutive sections of the same nodules as in the previous panels, followed by qRT-PCR analysis of indicated EMT genes. Results are shown as a heatmap of qRT-PCR results with values expressed as Log10 ratios of DNFGFR1- or DNTGFBR2-expressing HDFs versus controls. n (nodules per conditions) = 3.

Fig 3: Global Gene Expression Analysis Implicates ETV1 in FGF-TGF-ß Dualism(A) CAF effector genes upregulated by FGF2 versus TGF-ß1 treatment as per RNA-seq expression profiles of HDFs plus/minus FGF2 compared with published RNA-seq data of TGF-ß-treated HDFs (GEO: GSE79621). A complete list of up- and downregulated genes in FGF2-treated HDFs is shown in Table S1.(B) GSEA of expression profiles of DNFGFR1-expressing HDFs (left) or DNTGFBR2-expressing HDFs (right) versus controls against signatures of genes upregulated by TGF-ß1 or FGF2 treatment as identified in (A). The complete lists of genes is shown in Table S2.(C and D) Similar GSEA to that in (B) against the Hallmark gene set collection (Broad Institute). Shown is the positive versus negative enrichment of the EMT gene signature in DNFGFR1- versus DNTGFBR2-expressing HDFs (C) and the inverse pattern for the interferon alpha gene signature (D). A list of gene signatures positively associated with either type of HDF is shown in Figure S3F.(E) qRT-PCR analysis of ETV1 in HDFs treated with FGF2 or TGF-ß1 and HDFs expressing DNFGFR1 or DNTGFBR2. Values are expressed as Log10 ratios of treated HDFs versus controls. n (independent experiments) = 3, mean ± SEM, repeated ANOVA/Dunnett’s test, *p < 0.05, **p < 0.005.(F) Immunoblot analysis of ETV1 in HDFs plus/minus FGF2 or TGF-ß1 treatment. The membrane used in Figure 1F (top) was subsequently reprobed for ETV1 and GAPDH.(G) Immunofluorescence analysis of ETV1 (red) coupled with phalloidin staining (cyan) of HDFs treated as in (F). Shown are representative images (left) and quantification (right) of average ETV1 signal intensity per cell per field. Scale bar, 50 µm. n (fields per condition) = 15, mean ± SEM, one-way ANOVA/Dunnett’s test, **p < 0.005.(H and I) qRT-PCR analysis (H) and immunoblot (I) of ETV1 and indicated CAF effectors in ETV1-expressing HDFs versus controls. (H) Values are expressed as Log10 ratios over control. n (independent experiments) = 3, mean ± SEM, two-tailed paired t test, *p < 0.05, **p < 0.005, ***p < 0.001.(J and K) qRT-PCR analysis (J) and immunoblot (K) of ETV1 and indicated CAF effectors in HDFs infected with two ETV1-silencing lentiviruses (shETV1-3 and shETV1-5) versus empty-vector controls. (J) Values are expressed as Log10 ratios over control. n (independent experiments) = 3, mean ± SEM, two-tailed paired t test using the average of shETV1-3 and shETV1-5, *p < 0.05, **p < 0.005.(L and M) qRT-PCR analysis (L) and immunoblot (M) of ETV1 and indicated CAF effectors in HDFs infected with an ETV1-silencing lentivirus (shETV1-3) versus empty-vector control plus/minus FGF2 treatment. (L) n (independent experiments) = 3, mean ± SEM, two-tailed paired t test, *p < 0.05, **p < 0.005.(N and O) qRT-PCR analysis (N) and immunoblot (O) of ETV1 and indicated CAF effectors in ETV1-expressing HDFs versus controls plus/minus TGF-ß1 treatment. (N) n (independent experiments) = 4, mean ± SEM, two-tailed paired t test, **p < 0.005, ****p < 0.0001.

Fig 4: HDFs with Loss of Either FGF or TGF-ß Signaling Enhance Tumorigenic Expansion of Neighboring SCC Cells In Vivo(A) RFP-expressing CAL27 cells were admixed with either DNFGFR1- or DNTGFBR2-expressing HDFs versus controls, followed by parallel co-injection into mouse ears. Shown are representative images of injected ear pairs at day 20 after injection (left) and quantifications of relative red fluorescence intensity values normalized to day 1 values (right). Scale bar, 2 mm. DNFGFR1, n (injected ear pairs) = 16. DNTGFBR2, n (injected ear pairs) = 8. Mean ± SEM, two-tailed paired t test, *p < 0.05.(B) Similar tumorigenicity assays to the ones shown in (A) were performed with RFP-expressing CAL27 admixed with HDFs infected with an ETV1-silencing virus (left) or ETV1-expressing virus (right) versus empty-vector controls. Shown are quantifications of SCC expansion by in vivo fluorescence imaging as in (A). shETV1-3, n (injected ear pairs) = 6. ETV1 expression, n (injected ear pairs) = 8. Mean ± SEM, two-tailed paired t test, *p < 0.05.(C) H&E staining of parallel ear lesions formed by CAL27 cells co-injected with DNFGFR1-expressing HDFs versus controls. Scale bar, 500 µm.(D) Immunofluorescence analysis for pan-keratin (red) of lesions formed by CAL27 cells admixed with DNFGFR1- or DNTGFBR2-expressing HDFs. Shown are representative images (left) and quantifications expressed as number of pan-keratin-positive CAL27 cells per square millimeter (right). Scale bar, 100 µm. DNFGFR1, n (tumor pairs) = 4. DNTGFBR2, n (tumor pairs) = 3. Mean ± SEM, two-tailed paired t test, *p < 0.05, **p < 0.005.(E and F) Double immunofluorescence analysis for pan-keratin (red) and KI67 (green) of nodules formed by parallel back-skin injections of SCC13 cells admixed in Matrigel with DNFGFR1-expressing HDFs (E) or DNTGFBR2-expressing HDFs (F) versus controls. Shown are representative images (left panels), together with quantification of the fraction of KI67 and pan-keratin double-positive cells (right panels). Scale bar, 100 µm. n (nodules per condition, >800 cells per nodule, 4 fields per nodule) = 5, mean ± SEM, two-tailed paired t test, *p < 0.05.(G and H) Double immunofluorescence analysis of the same nodules in the (E) and (F) for pan-keratin (red) and keratin 10 (K10, green). Shown are representative images and quantification of the fraction of K10 and pan-keratin double-positive cells (left and right panels, respectively), with DNFGFR1-expressing (G) and DNTGFBR2-expressing (H) HDFs versus controls. Scale bar, 100 µm. n (nodules per condition, >700 cells per nodule, 4 fields per nodule) = 5, mean ± SEM, two-tailed paired t test, **p < 0.005, ***p < 0.001.(I and J) Double immunofluorescence analysis of the same nodules in the previous panels for pan-keratin (red) and CD31 (green). Shown are representative images and quantification of CD31-positive area with DNFGFR1-expressing (I) and DNTGFBR2-expressing (J) HDFs versus controls. Scale bar, 100 µm. n (nodules per condition, 4 fields per nodule) = 4, mean ± SEM, two-tailed paired t test, *p < 0.05, **p < 0.005.