Fig. 9: NOTCH1 silencing impairs cancer/stromal cell expansion in vivo.

a DsRed2-expressing SCC13 cells were admixed with CAFs (CAF14) plus/minus NOTCH1 silencing followed by parallel ear injections into NOD/SCID mice. Shown are images of two ear pairs 15 days after injection, quantification of red fluorescence signal (normalized to day 1 after injection) corresponding to SCC13 cell expansion, and tumor volumes (V = (length × width²) × 0.5). Statistical analysis of fluorescence intensity values over time course of the experiment was calculated by Two-way ANOVA, *p < 0.05. Tumor volume values are indicated as dots with mean ± s.d. two-tailed unpaired t-test, *p < 0.05. n(shCTR ear lesion) = 4, n(shNOTCH1 ear lesion) = 4. b Immunofluorescence analysis of ear lesions with antibodies against Ki67 (magenta) and human-specific VIMENTIN or KERATIN (cyan) for cell identification. Scale bar, 50 μm. Values for each ear lesion are indicated as dots with mean ± s.d. two-tailed unpaired t-test, ***p < 0.001. n(mice) = 4, n(shCTR ear lesion) = 4, n(shNOTCH1 ear lesion) = 4. Results from an independent experiment with a second CAF strain (CAF16) are in Supplementary Fig. 6a. c Immunofluorescence analysis of ear lesions with antibodies against the CAF marker PERIOSTIN (POSTN), macrophage (CD68), and endothelial (CD31) markers (magenta) with KERATIN (cyan). Scale bar, 50 μm. Values for each ear lesion are indicated as dots with mean ± s.d. two-tailed unpaired t-test, ***p < 0.001, ****p < 0.0001. n(shCTR ear lesion) = 4, n(shNOTCH1 ear lesion) = 4. Results from an independent experiment with a second CAF strain (CAF16) are in Supplementary Fig. 6a. d Immunofluorescence analysis of ear lesions with antibodies against the indicated proteins (magenta) together with human-specific anti-VIMENTIN antibodies for CAF identification (cyan). Scale bar, 50 μm. Values for each ear lesion are indicated as dots with mean ± s.d. two-tailed unpaired t-test, ***p < 0.001, ****p < 0.0001. n(shCTR ear lesion) = 4, n(shNOTCH1 ear lesion) = 4.