Fig. 3: ANKRD1 is required for cancer-associated fibroblast (CAF) maintenance.

a RT-qPCR of the indicated genes of patient-derived CAFs infected with ANKRD1-targeting (shANKRD1#1 and #2) and control shRNA (shCTRL), relative to shCTRL and expressed as amplification cycle thresholds normalized to RPLP0. n(strains) = 3, mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons test. b Immunofluorescence analysis of Ki67 in two strains of primary CAFs after ANKRD1 silencing, shown as the percentage of Ki67 positive cells per field. Number of fields for shCTRL in CAF1 (=27) and CAF2 (=27), for shANKRD1#1 in CAF1 (=24), and CAF2 (=28), for shANKRD1#2 in CAF1 (=20) and CAF2 (=27). Mean ± SD. One-way ANOVA with Dunnett’s multiple comparisons test. Ns=non-significant. c Growth enhancing activity of CAFs infected with ANKRD1-targeting (shANKRD1#1 and #2) and control shRNA (shCTRL) on neighboring SCC cells (FaDu) by co-culture assays. Immunofluorescence for Pan-keratin (Pan-KRT; for FaDu cells) and Vimentin (VIM; for CAFs). n(independent experiments) = 3, n(fields) for shCTRL, shANKRD1#1 and shANKRD1#2: 16 (Exp#1 and Exp#2), 10 (Exp#3). n(fields) for FaDu: 11 (Exp#1), 10 (Exp#2 and Exp#3), mean ± SD. One-way ANOVA with Holm-Šídák’s multiple comparisons test. Scale bar: 100 µM. d Representative phase contrast images of spheroid formation. CAFs infected with ANKRD1- targeting and control shRNA (shCTRL) were co-cultured with SCC13 cells on Matrigel-coated plates. Double IF analysis with anti-keratin and -vimentin antibodies. Mean ± SD, n(fields) = 20 (shCTRL), 18 (shANKRD1#1), 20 (shANKRD1#2), One-Way ANOVA with Dunnett’s multiple comparison’s test. Scale bar: 500 µm. Data point show the total fields imaged, from 4 independent experiments. e Organoid invasion assay of admixed CAFs infected with ANKRD1- targeting and control shRNA (shCTRL) and SCCs. Representative bright field images, and VIMENTIN and Pan-KRT IF analysis. Quantification of the invasion area was measured as the difference between the core and the invading area, delimited with dotted lines. n(fields) = 15 (shCTRL), 13 (shANKRD1#1), 12 (shANKRD1#2), 8 (Fadu), mean ± SD, One-way ANOVA with Dunnett’s multiple comparisons test. Scale bar: 200 µm. Data point show the total fields imaged, from 2 independent experiments. f Representative images of H&E staining of back lesions formed by FaDu cells co-injected with CAF#2 cells infected with either shANKRD1#1 or shCTRL vectors in contralateral mouse back skin. n(mice) = 5, mean ± SD. Orange dot: outlier identified by Grubbs test α < 0.1, two-tailed unpaired t-test, p = 0.0291. Scale bar: 500 µm, higher magnification: 100 µm. g Altered FaDu cell density and proliferation were quantified as the number of Pan-KRT positive cells per field. n(mice) = 5. Data points show the n(fields analyzed) = 25 for Control and n(fields analyzed) = 24 for shANKRD1. Mean ± SD, unpaired two-tailed t-test. p = 0.0003. Scale bar: 100 µm.