Figure 5.
Dll1 recruits CAFs to the TME in an IL-6 dependent manner during radioresistance and pharmacological blocking of Dll1 and IL-6 antibody sensitizes Dll1+ tumor cells to radiotherapy. A, Volcano plot from bulk mRNA-seq depicts enriched peaks for several upregulated genes in +TRT sorted Dll1+ tumor cells upon TRT. B, Gene set enrichment analysis (GSEA) demonstrates an enriched IL-6-JAK-STAT3 pathway signature in Dll1+ tumor cells post radiation (TRT). NES, normalized enrichment score and FDR, false discovery rate. (C and D), FACS plots (C) and ELISA (D) show higher IL-6 protein levels in Dll1+ tumor cells. E, Western blot showing activation of STAT-3 and p-STAT-3 in sorted CAFs from Dll1+ tumors after TRT (Data representative of minimum of two independent experiments). Please see Supplementary Fig. S13 for whole blots. F, Bar graphs demonstrate the total number of migrated CD140+/PDGFRα+ CAFs sorted from +TRT Py-Dll1+ tumors (Data representative of minimum of two independent experiments with two technical duplicates). G, A total number of 80,000 sorted live Py-Dll1+ tumor cells were transplanted in C57/B6 mice. Schematic model represents in vivo treatment of Dll1+ tumors with or without IgG control and anti-Dll1 antibody (18 mg/kg) and anti-IL-6 antibody (20 mg/kg) after a single dose of 6 Gy of TRT. H, Tumor growth curves of Dll1+ tumors from indicated treatments (n=6 tumors for IgG, n=8 tumors for Dll1Ab and combination group and n=10 tumors for IL-6 group). The black arrow indicates the day when TRT was given, and the blue arrow indicates the day when treatments started. I, Representative IF images, J, whole lung images and K, quantification show Dll1mCh+ and metastatic nodules (black arrows show nodules) in whole lungs (n=4 lungs/group). L, Scatter plot shows reduced CSC population in anti-Dll1 antibody or IL-6 treated tumors compared to IgG treated tumors. (M and N), Quantification and IF of αSMA staining and O, quantification of FAP staining in +TRT Dll1+ tumors upon indicated in vivo treatments. The dots in scatter plot represents field of views (FOVs), n=4 tumors/group. Data are presented as the mean ± SEM. Two-way ANOVA with Bonferroni post-test (H), One-way ANOVA with TUKEY post-test (F, K-M and O) were used to calculate p values. *p<0.05, ** p<0.01; **** p<0.0001. Scale bars, 4 mm (I and J) and 100 μm (N).