Fig 2: KISS1R is a prognostic factor and a potential therapeutic target in advanced TCs.A (left panel) H&E analysis of primary tumor (P), lymphnodes (Ly) and lung (Lu) metastatic lesions, generated by orthotopic injection of D22 TPCs engineered with the indicated mutations. Scale bars, 100 µm. (right panel) Immunohistochemical analysis of TIMP1, MMP9, CD44, ß-catenin, TWIST and SNAIL on primary and metastasis mouse avatars. Ctrl= negative control. Scale bars, 100 µm. B Immunohistochemical analysis of TIMP1, MMP9, CD44, ß-catenin, TWIST and SNAIL on primary and metastatic PTC (ID#61) and ATC (ID#96) patient-derived tumors harboring BRAF/TP53 mutations. Ctrl= negative control. Scale bars, 100 µm. For (A and B) n = 5 tissues analyzed. c Heatmap of Wnt-, EMT- stemness and metastasis-related genes (2-?Ct expression values) in primary (P), metastatic (M) tumor xenografts and primary (P) and metastatic (M) PTC- and ATC-patient derived tumors, harboring BRAF/TP53 mutations. Data are presented as normalized mRNA expression values of three independent experiments. D Venn diagram showing common upregulated genes with logFC > 3.5 in metastasis (M) versus primary (P) tumor xenografts, in PTC-derived metastasis (PTC M) versus primary tumors (PTC P) and in ATC primary versus PTC primary tumors. E and F, (left panels) Immunohistochemical analysis of KISS1 (E) and KISS1R (F) on primary and metastatic PTC (ID#61) and ATC (ID#96) patient-derived tumors harboring BRAF/TP53 mutations. n = 5 tissues analyzed. Scale bars, 100 µm. (right panels) R2 database analysis of KISS1 (E) and KISS1R (F) mRNA expression levels in normal thyroid (nt, n = 6), PTC (n = 6), FTC (n = 6) and ATC (n = 10) patient-derived tissues (R2 database Tumor Thyroid Carcinoma all - Huettelmaier - 28 - tmm - jbseqrnanb1). Boxes represent the IQR and midline represents the median. Statistical significance was calculated using ANOVA test. G Immunohistochemistry score analysis of KISS1R expression in normal thyroid tissue (n = 56), primary PTC tumors (PTC P, n = 73), PTC-derived loco-regional lymphnode metastasis (PTC M, n = 19) and primary ATC tumors (n = 5). Mutational status of 97 primary and metastatic human tissues is reported in Supplementary Data 2. Boxes represent the IQR and midline represents the median. Statistical significance was calculated using Kruskal-Wallis test.

Fig 3: KISS1R and TIMP1 targeting restore the functional iodine uptake by increasing NIS expression.A Relative mRNA expression levels of PAX8, TG, TSHR, TPO and TTF1 in the indicated engineered D22 treated with KISS1R inhibitor (KISS1Ri) alone or in combination with TIMP1 inhibitor (TIMP1i) for 24 h. Data are presented as fold change over vehicle ± SD of three independent experiments. B and c Immunofluorescence analysis of NIS and cell positivity in D22 TPCs engineered with the indicated mutations and treated as in (A) for 72 h. Nuclei were counterstained by Toto-3. Statistical significance was calculated using the unpaired two-tailed t test. Scale bars, 20 µm. Data are mean ± SD of 3 independent experiments. n = 12 wells analyzed. D Relative mRNA expression levels of NIS in cells engineered and treated as in (A) for 24 h. Data are presented as fold change over vehicle ± SD of three independent experiments. E and F Immunoblot of NIS (E) and relative optical density ratio (F) in cells as in (A) for 48 h. ß-actin was used as loading control. One representative of three independent experiments is shown. Source data are provided as a Source Data file. G Radioiodine uptake in the indicated established thyroid cancer cell lines and engineered D22 TPCs treated as in (A) for 48 h. For (F and G) statistical significance was calculated using the two-tailed unpaired t test and data are mean ± standard error of three independent experiments. H (left panel) Growth kinetics of xenograft tumors generated by subcutaneous injection of D22 TPCs engineered for NRASQ61R/TP53R248Q treated with TIMP1 inhibitor (TIMP1i) and KISS1R inhibitor (KISS1Ri) alone and in combination. Statistical significance was calculated using the unpaired two-tailed t test. Data are shown as mean ± SD. n = 6 mice per group. (right panels) Immunohistochemical analysis of NIS on xenograft tumors obtained following injection of D22 TPCs engineered for NRASQ61R/TP53R248Q treated as indicated, left panel. Scale bars, 100 µm. I Kaplan-Meier graph showing the murine survival of D22 TPCs engineered with NRASQ61R/TP53R248Q treated as in (H). The statistical significance between groups was evaluated using a log rank Mantel-Cox test.