Fig 1: FRC cell responses to irradiation are dependent on TNC expression.(A) Representative Scanning Electron Microscopy (SEM) images of irradiated (IR) or non-irradiated (NIR) WT and TNCKO FRCs. Scale bar, 50 µm. (B) Cell density (analysis of 100 SEM images for each condition) of WT and TNCKO FRCs expressed as a ratio of IR to NIR cells. Mean ± SD; Student T test, ****P < 0.0001. (C) GSEA map display of differentially expressed protein categories in WT or TNCKO FRCs after irradiation. The color code indicates the adjusted P value range, and the black circles the approximate number of proteins that belong to each GO term. (D) Forward (FWD) E-Modulus average (kPA) of the corresponding Atomic Force Microscopy measurement of the CDM of WT or TNCKO FRCs before (NIR) or after irradiation (IR). N = 3. Mean ± SEM (E, F) Representative IF images of DC2.4 cells adherent on the CDM from the FRCs (WT or KO) after the chemoretention assay. DC2.4 nuclei are stained with DAPI (arrows point at the nuclei) and were counted on the CDM in (F) upon migration toward CCL21, pre-treatment with the anti-CCR7 neutralizing antibody (αCCR7), the TNC inhibitory nanobody Nb3, or no treatment control. Scale bar, 200 µm. Mean ± SEM; n = 6 per condition; Kruskal–Wallis test and Dunn post-test, *P < 0.05, **P < 0.01, ***P < 0.005. (G, H) Expression of Ccl21 (G) and Tnc (H) was determined by qRTPCR after treatment of FRCs (WT, KO, NIR, IR) with TGFβ (10 ng/mL for 24 h) or with the CM from the OSCC13 cells in the presence or absence of GW788388 (GW). Mean ± SEM; n = 6–9 per condition; Kruskal–Wallis test and Dunn post-test, *P < 0.05, **P < 0.01. The exact P values are listed in Appendix Table S5. Source data are available online for this figure.
Fig 2: Expression of TNC impacts tumor radiosensitivity, and immune cell infiltration in the TdLNs of a murine model of OSCC.(A) Quantification of the tongue tumor size in 4NQO-treated WT and TNCKO (KO) mice non-irradiated (NIR) or irradiated (IR), n = 5 per group. (B) Representative IF images for TNC and the proliferation marker Ki67 in NIR and IR 4NQO tumors of WT and TNCKO mice (T, tumor islet, S, Stroma). Scale bar, 200 µm. (C) Quantification of Ki67-positive cells (%) in the tumor per image. Four images per tumor, n = 5 mice per group. (D, E) Quantification of the immunostaining signal to evaluate the spatial distribution of the CD45+ (D) and CD11c+ (E) cells present in the tumor nest as a ratio of positive cells over the total of cells per image. (F, G) FACS analysis of CCR7+ dendritic cells (DC) expressed as a ratio (%) of the total DC population (CD45 + , CD11c + , MHCII + ) (F) and FRCs (GP38 + , CK10-, CD31−) expressed as a ratio (%) of the total CD45-negative cells (G). (H) ELISA for CCL21 in NIR and IR 4NQO tumors of WT and TNCKO mice. N = 5–6 per group. (I) Volcano plot of deregulated genes, after RNA sequencing of the TdLN of WT and TNC KO mice. N = 3 WT and 3 TNCKO TdLN. (J, K) Representative IF images for CD3 and B220 in the TdLN of WT or TNC KO mice (J) and quantification of their intensity/area in n = 2–4 sections of N = 5 TdLNs per group (K). Scale bar, 50 μm. Mean ± SEM; Kruskal–Wallis test and Dunn post-test, *P < 0.05, **P < 0.01, ***P < 0.005. The exact P values are listed in Appendix Table S5. Source data are available online for this figure.
Fig 3: The FRC-OSCC crosstalk impacts irradiation responses in a TNC-dependent manner.(A) immunofluorescence images of OSCC13 (OSCC) and FRC (WT, shC, TNCKO) after coculture in a 2:1 ratio. Scale bar, 200 µm. (B) Gene expression (qRTPCR) analysis of Ccl21 expression in the cocultured FRCs after MACS, before or 2 days after exposure to 10 Gy IR. (C, D) GP38 and Rad51 immunofluorescence images of the cocultured OSCC and FRC (shC, shTNC,TNCKO) cells before or 2 days after exposure to 10 Gy IR (C) and quantification (%) of Rad51 positive OSCC cells (D). Arrows indicate the OSCC cells expressing Rad51. Scale bar, 200 µm. (E, F) qRTPCR analysis of Tgfb1 (E) and Twist (F), in the cocultured OSCCs before or 2 days after exposure to 10 Gy IR. (G) Immunofluorescence images of OSCCs and FRCs, cocultured in a 2:1 ratio for a total of 4 days, 2 days prior to and 2 days after exposure to 10 Gy IR, under GW treatment for the indicated molecules. Arrows indicate the OSCC cells that undergo EMT. (H, I) Signal quantification in the OSCC cells expressed as % of all tumor cells. Scale bar, 50 µm, N = 3 experiments; Error bars represent mean ± STDEV; ordinary one-way ANOVA test, **P < 0.01. ***P < 0.005. ****P < 0.0001. The exact P values are listed on Appendix Table S5. Source data are available online for this figure.
Fig 4: TNC expression plays a pivotal role in defining the FRC phenotype.(A) Representative IF images for GP38 (podoplanin) and CCL21 in non-irradiated (NIR) FRCs isolated from lymph nodes of naïve WT or TNCKO mice. The same microscopic field of the image showing DAPI (blue) and GP38 (red) in the TNCKO FRCs was co-stained for the marker VCAM1 (green), which is displayed separately in Fig. EV2A. Scale bar, 50 µm. (B) Display of differentially expressed proteins in WT and TNCKO FRCs applying the GSEA analysis tool. The color code indicates the adjusted P value range and the black circles the approximate number of proteins that belong to each GO term. RNA seq gene expression analysis of WT and TNCKO FRCs represented as heatmaps for deregulated genes according to FRC markers (C) and chemokine and cytokine molecules (D). Bold text indicates genes with established roles in the respective category that are discussed in the text. (E) Representative image of Col XII, TNC, FN, and Col IV expression in the CDM of WT or TNCKO FRCs. Scale bar, 200 µm. (F) Quantification of CCL21 by ELISA in WT or TNCKO FRCs, either non-treated control (Ctrl) or treated with soluble TNC (10 µg/mL) for 24 h. N = 4 experiments. Mean ± SD, Kruskal–Wallis test and Dunn post-test, *P < 0.05, **P < 0.01 ***P < 0.001. The exact P values are listed in Appendix Table S5. Source data are available online for this figure.
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