Cancer-associated fibroblast-derived periostin promotes papillary thyroid tumor growth through integrin-FAK-STAT3 signaling

Figure 1. Expression of POSTN in human and mouse papillary thyroid tumors and its clinical prevalence. (A) Representative images of the immunohistochemical staining of POSTN in human papillary thyroid tumor and adjacent normal tissue. Scale bars, 50 μm. (B and C) Western blot (B) and qRT-PCR (C) analyses of POSTN in the tissues of human papillary thyroid tumors (T) and adjacent normal tissues (N) (Student's t test, n = 5). (D) GEPIA database analysis of POSTN expression in human normal thyroid (n = 59) and thyroid tumors (n = 512). Transcripts per million (TPM). Student's t test. (E) GEPIA database stage-plot analysis of the relationship between POSTN expression and tumor stages in thyroid cancer (χ2 test). (F) GEPIA database correlation analysis of overall survival with POSTN levels in thyroid cancer (Low POSTN group, n = 459; high POSTN group, n = 128, log rank test). (G-I) Immunohistochemical staining (G), western blot (H) and qRT-PCR (I) analyses of POSTN in thyroid tissues from mice after the orthotopic injection of IHH-4 cells or PBS (Student's t test, n = 3). Scale bars, 50 μm. Data are shown as means ± SEM. *, P < 0.05; **, P < 0.01.

Figure 2. POSTN is mainly derived from CAFs in mouse papillary thyroid tumors. (A) Representative immunofluorescence co-staining of POSTN with α-SMA, EpCAM, CD31 or F4/80 in orthotopic mouse thyroid tumors. Nuclei were counterstained with DAPI. Scale bars, 50 μm. (B) Plots of fluorescent intensity along the yellow line of images in (A). (C) Representative immunohistochemical staining of POSTN, α-SMA and EpCAM in the orthotopic thyroid tumors. Scale bars, 50 μm. (D) Fluorescence in situ hybridization of Postn and Acta2 in orthotopic thyroid tumors. Nuclei were counterstained with DAPI. Scale bars, 50 μm. (E) Western blot analysis of POSTN expression in Nthy-ori-3-1, IHH-4 and TPC-1 cells. rhPOSTN was used as a positive control. (F and G) POSTN protein (F) and mRNA expression (G) in isolated CAFs and tumor cells (Student's t test, n = 3). rhPOSTN was used as a positive control. Data are shown as means ± SEM. **, P < 0.01.

Figure 3. POSTN deficiency inhibits tumor growth in vivo, whereas POSTN promotes IHH-4 cell growth in vivo and in vitro. (A and B) Bioluminescence imaging (A) and quantification of tumor burden (B) in Postn+/+Rag1-/- (Postn+/+) and Postn-/-Rag1-/- (Postn-/-) mice after orthotopic injection with IHH-4 cells (n = 6). (C and D) Images (C) and quantification of weights (D) of isolated orthotopic tumors from mice in each group in (A) (n = 6). (E and F) Immunohistochemical staining for Ki67 (E) and quantitative analysis of Ki67+ cells (F) in tumor tissues. Scale bars, 25 μm (n = 3). (G) Western blot analysis of POSTN and Cyclin D1 in orthotopic tumor tissues from Postn+/+Rag1-/- and Postn-/-Rag1-/- mice. (H) qRT-PCR analysis of Cyclin D1 (Ccnd1) in orthotopic tumor tissues from Postn+/+Rag1-/- and Postn-/-Rag1-/- mice (n = 4). (I and J) Bioluminescence imaging (I) and quantification of tumor burden (J) in Postn-/-Rag1-/- mice after orthotopic injection with IHH-4 cells transfected with POSTN or control vector (n = 6). (K and L) Images (K) and quantification of weights (L) of isolated orthotopic tumors from mice in each group in (I) (n = 6). (M and N) Immunohistochemical staining for Ki67 (M) and…

Figure 4. Deficiency of POSTN in CAFs impairs CAF-promoted papillary thyroid tumor growth in vivo and IHH-4 cell proliferation in vitro. (A and B) Bioluminescence imaging (A) and quantification of tumor burden (B) of Postn-/-Rag1-/- mice after orthotopic injection with IHH-4 cells alone or together with WT or KO CAFs (n = 6). (C and D) Images (C) and quantification of weights (D) of isolated orthotopic tumors from mice in each group in (A) (n = 6). (E and F) Immunohistochemical staining for Ki67 (E) and quantitation of Ki67+ cells (F) in tumor tissues from each group in (A). Scale bars, 25 μm (n = 6). (G and H) Immunofluorescence images (G) and quantitation (H) of Ki67+ IHH-4 cells cultured alone or co-cultured with WT or KO CAFs (n = 6). (I) Western blot analysis of POSTN in human CAFs transfected with POSTN or control vector. (J and K) Immunofluorescence images (J) and quantitation (K) of Ki67+ IHH-4 cells co-cultured with the vector control or POSTN-overexpressing human CAFs (n = 4). Data are shown as means ± SEM. One-way ANOVA (B, D, F and H). Student's t test (K). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., no significant difference.

Figure 5. POSTN promotes the proliferation and IL-4 expression in IHH-4 cells by activating the integrin-FAK-STAT3 signaling. (A) Immunohistochemical analysis of the expression of IL-4 in tumor tissues from Postn+/+Rag1-/- and Postn-/-Rag1-/- mice. Scale bars, 50 μm. (B-D) Western blot (B), qRT-PCR (C) (n = 4) and ELISA (D) (n = 6) analyses of IL-4 expression in tumor tissues of Postn+/+Rag1-/- and Postn-/-Rag1-/- mice. (E) Immunohistochemical analysis of the expression of IL-4 and EpCAM in human papillary thyroid tumor. Scale bars, 50 μm. (F and G) Western blot (F) and qRT-PCR (G) analyses of IL-4 expression in IHH-4 cells cultured alone or co-cultured indirectly with WT or KO CAFs (n = 3). (H and I) Western blot (H) and ELISA (I) analyses of IL-4 level in IHH-4 cells treated with or without 100 ng/mL rhPOSTN (n = 6). (J and K) Western blot (J) and qRT-PCR (K) analyses of the indicated proteins and genes in IHH-4 cells treated with 100 ng/mL rhPOSTN in combination with antibodies against integrins αvβ3 or αvβ5 (n = 4). (L) Analysis of the proliferation of IHH-4 cells treated as in (J) for the indicated time by CCK8 assay (n = 4). (M and N) Western blot (M) and qRT-PCR (N) analyses of the…

Figure 6. IL-4 enhances the activation and POSTN expression in CAFs. (A-C) Western blot (A) and qRT-PCR (B and C) analyses of POSTN and Acta2 in CAFs cultured alone or co-cultured indirectly with IHH-4 or TPC-1 cells (n = 4). (D and E) Relative mRNA levels of Postn (D) and Acta2 (E) in isolated CAFs treated with rmIL-4 for the indicated time (n = 4). (F and G) Relative mRNA levels of Postn (F) and Acta2 (G) in isolated CAFs treated with rmIL-4 for the indicated doses for 24 hours (n = 3). (H and I) Western blot (H) and qRT-PCR (I) analyses of POSTN in CAFs treated with rmIL-4 (n = 3). (J) ELISA analysis of POSTN level in CAFs treated with rmIL-4 (n = 6). (K and L) Immunofluorescence images (K) and quantitation (L) of POSTN+ cells in CAFs treated with or without rmIL-4. Scale bars, 50 μm (n = 6). (M-O) Western blot (M and N) and qRT-PCR (O) analyses of indicated proteins and Postn gene in CAFs treated with rmIL-4 alone or together with indicated inhibitors (n = 3). (P) Schematic representation of full length and two truncated luciferase reporters of mouse Postn promoter, and predicted binding sequences were predicted through a website-based search (https://guolab.wchscu.cn/hTFtarget/). (Q)…

Figure 7. The correlation between POSTN and IL-4 expression in human thyroid tumors. (A and B) The expression of POSTN and IL-4 in normal thyroid tissues (NT, n = 29), papillary thyroid cancer [PTC, n = 46] and anaplastic thyroid cancer [ATC, n = 10] was analyzed in the GSE33630 dataset. One-way ANOVA with multiple comparisons test. (C) Spearman correlation analysis of IL-4 with POSTN of human papillary thyroid cancer in the GSE33630 database (n = 46). Spearman correlation coefficient (R). (D) Spearman correlation analysis of POSTN expression with infiltration level of CAF of human thyroid cancer in the Timer 2.0 database. (E) Spearman correlation analysis of POSTN with STAT3 of human thyroid cancer in the GEPIA database. Normalized by HMBS. (F) Spearman correlation analysis of IL-4 with STAT6 of human thyroid cancer in the GEPIA database. Normalized by HMBS. (G and H) Western blot (G) and qRT-PCR (H) analyses of POSTN and IL-4 in clinical papillary thyroid tumors (n = 3). Two-way ANOVA with multiple comparisons test. (I) Representative immunohistochemical images of POSTN and IL-4 in clinical papillary thyroid tumors and their corresponding adjacent tissues. Scale bars, 50 μm. (J) Model…