Extended Data Fig. 1: Transcriptomic analyses of Mdk-depleted melanoma and effect of MDK on DCs.
(a) Summary of the workflow followed to identify MDK-associated gene signatures to then define their impact on mouse models and patient-derived datasets. (b) Significantly dysregulated processes (GO Term) of upregulated genes (n = 362) defined by RNAseq in B16.R2L-shMdk (n = 4) vs shCtrl (n = 4) independent tumor allografts. Data were analyzed using Cytoscape (v3.10.1) and ClueGo (v2.5.10). Statistical analysis included correction with Bonferroni step down. (c) Normalized Enrichment Scores (NES) of the indicated gene ontology (GO) terms defined by GSEA in B16.R2L-shMdk (n = 4) vs shCtrl (n = 4) independent allografts: Innate Immune Responses (GO:0045087), T Differentiation (GO:0030217), Myeloid and Leukocyte Differentiation (GO:0002573) and DC activation (GO:0001773). (d) Representative GSEA plots for GO terms Antigen processing and presentation (GO:0019882), DC differentiation (GO:0097028) enriched in B16.R2L-shMdk (n = 4) vs the shCtrl (n = 4) allografts. (e) Gating strategy used to identify cDCs, cDC1s and cDC2s from FLT3LG-BMDCs cultures. (f) Pie charts from FLT3LG-BMDCs cultures treated with or without rMDK and analyzed as in panel Fig. 1d (n = 4 independent allografts). (g) Representative flow cytometry plots and the corresponding quantification (h) of cell viability of FLT3LG-BMDCs in the absence or presence of rMDK (n = 4 independent mice per condition). Data represented as mean +/- SEM. Unpaired two-tailed t-test was used to assess statistical significance. (i) RT-qPCR to assess the expression of cDC1-associated genes (Batf3, Irf8 and Nfil3) in marrow cells cultured with 50 ng/ml of FLT3LG in the absence (Control, n = 3) or in the presence of murine rMDK (10 ng/ml, n = 3) during the first three days of the differentiation. Data represented as mean +/- SEM Statistical significance was determined by two-way ANOVA corrected for Sidak’s multiple comparisons. Data correspond to BMDCs isolated from 3 independent mice. (j) Amount of MDK (ng/ml) secreted by different melanoma cell lines used in the gain-of-function analyses for B16.F10, B16.F1, YUMM1.1, YUMM2.1 and WM164. Sample size n = 3 independent biological replicates for all groups. Data represented as mean +/- SEM. Statistical significance was determined by one-way ANOVA with Brown-Forsythe test. p-value: **** = <0.0001. (k) Percentage of BMDCs obtained from FLT3LG-BMDCs cultured with conditioned media from B16.F10 (n = 4), YUMM1.1 (n = 5) and YUMM2.1 (n = 5) overexpressing MDK or isogenic control (NEG). Statistical significance was determined by an unpaired two-tailed t-test. All violin plots show median values (center dash-line), quartiles (dotted-lines) and minimum and maximum values (edges of the violin), with individual values plotted. Data correspond to BMDC cultures from independent mice. (l) Amount of MDK (ng/ml) secreted by B16.R2L cell line used in loss-of-function analyses with shRNA and siRNA. Sample size: B16.R2L-shCtrl n = 3; -shMdk n = 4; -siCtrl n = 3; -siMdk #34 n = 8; -siMdk #35 n = 8; -siMdk #36 n = 8. Data represented as mean +/- SEM of independent biological replicates. Statistical significance was determined by one-way ANOVA with Brown-Forsythe test. (m) mRNA expression of the indicated cDC1-associated genes in MutuDC1940s cultured with conditioned media from B16.R2L-shMdk (n = 3) relative to -shCtrl (dash line, n = 5). Data represented as mean +/- SEM. Statistical significance was determined by two-way ANOVA corrected for Sidak’s multiple comparisons (p-value: **** = <0.0001, *** = 0.0005). Data correspond to independent experiments. (n) mRNA expression of cDC1-associated genes in marrow cells cultured with FLT3LG (50 ng/ml) treated with conditioned media from B16.R2L-siMdk#34 and -siMdk#35 relative to -siCtrl (dash line), during the first three days of the differentiation. Data correspond to three technical replicates from two independent experiments. All values are plotted. (o) Percentage cDC1s obtained from FLT3LG-BMDCs cultured with non-tumoral conditioned media (Non-TCM, n = 3), conditioned media from B16.R2L-siCtrl (n = 3) or -siMdk#36 (n = 3). Data correspond to BMDC cultures from three independent mice per group. Statistical significance was determined by one-way ANOVA corrected for multiple comparisons (Tukey test). All plots show median values (center line) with individual values plotted.
