CD271 expression in cSCC biopsies and spheroids a Hematoxylin and Eosin (H&E) staining of AK, in situ, WD and MD/PD cSCC biopsies. CD271, Cyclin D1, TrkA, KRT1 and KRT13 expression evaluated by immunohistochemistry or immunofluorescence. Scale bar 100 µm. b Immunohistochemistry and immunofluorescence staining scored as follow: 0 (no cells; positivity to 20% of cells positivity), 0.1–0.75 (30–50% of cells positivity; 1–1.5 (60% to 100% of cells positivity). c Graphical representation of the KRT13/KRT1 intensity ratio. d Representative pictures of in situ, WD, and MD/PD spheroids. Scale bar = 100 μm. e Spheroid total area measured by ImageJ software and f spheroid viability evaluated by MTT assay. g Expression of E-cadherin, KRT13, and KRT10 evaluated in patient-derived spheroids by western blotting. β-actin was used as control. h Graphical representation of KRT13 and KRT10 relative protein expression ratio. i NTs and NTRs mRNA expression evaluated in patient-derived spheroids by qPCR. Heatmap created by Prism Graph pad software. β-actin was used as a housekeeping gene. For all experiments, the results are shown as mean ± SD of three independent experiments. Statistical analysis was performed using the two-way ANOVA. *0.01 < p < 0.05, ***0.0001 < p < 0.001, ****p < 0.0001

CD271 modulation affects cSCC spheroid phenotype a Representative picture of in situ, WD, or MD/PD patient-derived CD271-transduced or mock spheroids. Scale bar = 100 μm b CD271 expression by qPCR. β-actin was used as housekeeping gene. c Spheroid total area measured by ImageJ software. d Spheroid viability evaluated by MTT assay. e E-cadherin, KRT10, and Slug expression evaluated in patient-derived spheroids by western blotting. Tubulin was used as loading control. f Representative picture of SCC13 spheroids treated as described. Scale bar = 100 μm g Spheroid total area measured by ImageJ software. h Spheroid viability evaluated by MTT assay. i CD271, E-cadherin, KRT10, ERK1/2, pERK1/2, and Slug expression evaluated in CD271-overexpressing or silenced SCC13 spheroids by western blotting. pERK1/2/ERK1/2 expression ratio analyzed by ImageJ software. β-actin was used as loading control. j GO Biological Process term fold-Enrichment of RNA-seq data of CD271-transduced SCC13 spheroids vs mock determined by PANTHER Tools (a triplicate for n = 80 mock or CD271 spheroids). For all experiments, the results are shown as mean ± SD of three independent experiments. Statistical analysis was performed using the two-way ANOVA or multiparametric T-test. *0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, ****p < 0.0001

CD271 overexpression promotes cSCC differentiation a CD271 and mock spheroids were fixed with 4% PFA and embedded in paraffin. Spheroid histology was evaluated by Hematoxylin and Eosin (H&E) staining (scale bar = 50 μm) and b the number of nuclei was measured by ImageJ software. c Density, Weight, and Diameter of SCC13-Spheroids measured by W8™. A minimum of 10 single spheroids was analyzed for each test condition and values were extrapolated from at least 10 repetitions. d The expression of CD271, TrkA, KRT1 and Ki67 were evaluated in mock vs CD271-overexpressing SCC13-spheroids by immunofluorescence. Nuclei were stained with DAPI. e Left panel: Viability and apoptosis were measured in CD271 vs mock spheroids by LIVE/DEAD® assay (Calcein: Green, Ethidium Bromide Red). Right panel: percentage of Ethidium Bromide positive cells determined by ImageJ software analysis of micrographs. Scale bar = 50 μm f CD271 and mock spheroids implanted into collagen I and dermal fibroblast matrix and monitored for 2 weeks (scale bar = 100 μm). % of fragmentation and invasion area was determined by ImageJ software analysis. g H&E and CD271, KRT10, and KRT15 expression evaluated by immunofluorescence of mock and CD271-overexpressing SCC13-derived skin reconstruct. Nuclei were stained with DAPI. Statistical analysis was performed using the two-way ANOVA and multiparametric T-test. *0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, ****p < 0.0001. Scale bar = 50 µm

CD271 modulation improves PDT or chemotherapy efficiency a Schematic representation of photodynamic therapy (PDT) on SCC spheroids. b Cell viability evaluated at 24 h in mock vs CD271-overexpressing SCC13-derived spheroids treated with PDT for 15 min, 1 h or 3 h (n = 3). c Left panel: Spheroids total area, corresponding to 3 h PDT treatment, evaluated at 24 h. Right panel: representative brightfield images and ImageJ mask of mock and CD271-overexpressing spheroids d Representative images for PI staining (red)/brightfield of mock vs CD271 spheroids after treatment with 5FU (1, 10 and 50 μg/ml) or DMSO (control) at 48 h. Scale bar = 100 μm e Histogram representing PI staining/total spheroid area ratio (for each condition, n = 3). Results are shown as mean ± SD. Statistical analysis was performed using the two-way ANOVA and multiparametric t-test. *0.01 < p < 0.05, ***0.0001 < p < 0.001, ****p < 0.0001

Effects of CD271 activation on cSCC spheroid viability and invasion a Cell viability (MTT assay) and b total area of SCC13 spheroids (n≥6) after β-Amyloid (40 μM and 60 μM) treatment as compared to control (PBS). c Left panel: Propidium Iodide (PI) staining (red)/brightfield on mock vs CD271-overexpressing SCC13 spheroids after β-Amyloid treatment as compared to control (PBS) at 48 h. Right: Bar chart representing PI-stained area (Pixel) (n = 3). Scale bar = 50 μm dNGFR, BIRC5 and CXCL8 mRNA levels (n = 8). e Left panel: Representative images of SCC13-derived implanted spheroid treated with β-Amyloid or PBS (control) and monitored for 2 weeks (scale bar = 50 μm). Middle panel: total invasion area. Right panel: percentage of fragmentation. f Left panel: PI staining/brightfield of CD271 and mock SCC13 spheroids treated with K252 (200 nM) or DMSO at 48 h. Right panel: Quantification of the PI staining/Total area ratio. Scale bar = 50 μm g Total area of SCC13 spheroids treated with TrkA/Fc (2 µg/ml), βAmyloid (40 μM) or the combinations TrkA/FC/βAmyloid (n = 3). Results are shown as mean ± SD of three independent experiments. Statistical analysis was performed using the two-way ANOVA and multiparametric T-test. *0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, ****p < 0.0001

CD271 activation increases response to chemotherapy and promotes macrophage recruitment in zebrafish a CD271 expression in SCC13 cells treated with Cisplatin (5μg/mL) vs control by FACS. b Representative images of SCC13 cells (red) injected into zebrafish at 7dpi after treatments as described. Scale bar = 500µm. c cSCC metastasis quantified and classified as Full metastases, Initial metastases, or In place. d-e Quantification of the red fluorescence intensity of tumor mass in zebrafish by ImageJ software and ImageJ mask. f Left images: schematic representation of mpeg1 positive cells (red) in zebrafish injected with mock or CD271-overexpressing cells (green) within the analyzed area (blue). Right images: schematic representation of treatments. g Quantification of mpeg1-positive cells in zebrafish injected with mock or CD271-overexpressing cells and h in zebrafish injected with SCC13 cells after treatments as described, by whole-mount in situ hybridization. PBS was used as a control (n = 3, 3 independent evaluations). Results are shown as the mean percentage of three independent experiments (minimum of 20 injected zebrafish/condition). Statistical analysis was performed using the two-way ANOVA and multiparametric T-test. *0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, ****p < 0.0001

CD271 expression increases the immune cell recruitment in vivo and in vitro a Representative images of mock and CD271-overexpressing SCC13 cells (green) injected into WT (indicated as WT/SCC13 in the figure) or Tg(lysC:DcRed)nz50 (indicated as LysC/SCC13) zebrafish with or without the simultaneous treatment with the L-LME. In the Tg(lysC:DcRed2)nz50 zebrafish, leucocytes possess a red fluorescence. Scale bar = 500 µm. b and c cSCC metastases quantified and classified as Full metastases, Initial metastases, or In place. d Quantification of LysC-positive cells in zebrafish injected with mock or CD271-overexpressing cells e SCC13 cells were stained with Vybrant DIO Cell-Labeling Solution (green) and seeded in a 96-well plate coated with 1,5% of agar. cSCC spheroids were transduced by infection with mock or CD271 viral vectors. After 72 h, THP-1 cells were stained with Vybrant DII Cell-Labeling Solution (red) and co-cultured with cSCC spheroids. Pictures were made after 24 and 72 h from the THP-1 seeding. Scale bar = 100µm. f The invasion area of THP-1 was measured by ImageJ software. Results are shown as the mean percentage of three independent experiments (minimum of 20 injected zebrafish/condition). Statistical analysis was performed using the two-way ANOVA and multiparametric T-test. **0.001 < p < 0.01, ****p < 0.0001

CD271 expression abolishes cSCC metastasis in zebrafish a Above panel: Zebrafish injected with mock or CD271-overexpressing fluorescent stained SCC13 cells (red; about 50 cells/embryo) at 5 dpi (day post-injection). Scale bar = 500 µm. Bottom panel: Metastases were quantified and classified as Full metastases, Initial metastases, and In place. b Above panel: Representative detail of fluorescent cSCC cells (red) and ImageJ mask. Bottom panel: Quantification of the red fluorescent intensity of cell mass in zebrafish by ImageJ software. c Expression of CD271, E-Cadherin, ERK1/2, pERK1/2, p-p38, Slug and Snail in cell injected into zebrafish. βactin was used as loading control. d Above panel: Zebrafish injected with scramble or CD271-silenced fluorescent stained SCC13 cells (red; about 50 cells/embryo) at 5 dpi (day post-injection). Scale bar = 500 µm. Bottom panel: Metastasis quantification as in (a). e Above panel: Representative detail of fluorescent cSCC cells in red and ImageJ mask. Bottom panel: quantification of the red fluorescent intensity of cell mass in zebrafish by ImageJ software. f Expression of CD271, E-Cadherin, ERK1/2, pERK1/2, p-p38, Slug and Snail in cell injected into zebrafish. βactin was used as loading control. Results are shown as the mean percentage of three independent experiments and a minimum of 20 injected zebrafish for each condition was used. Statistical analysis was performed using the two-way ANOVA and multiparametric T-test. *0.01 < p < 0.05, ***0.0001 < p < 0.001, ****p < 0.0001