Figure 2.

Lack of Tumor Revascularization during Resistance against Nintedanib Therapy

(A and B) Microvessel densities (A) and CD31-positive area fractions (B) were quantified in Py2T tumors from mice treated for 1 week (ST) or 3 weeks (LT) with vehicle or nintedanib.

(C) Endothelial cell apoptosis (CD31, green; cCasp3, red) is shown on representative immunofluorescence picture of a tumor from a 1-week (ST) nintedanib-treated mouse. DAPI was used to visualize cell nuclei. The scale represents 20 μm.

(D) Quantification of endothelial cell apoptosis by immunofluorescence co-staining for cCasp3 and CD31 in tumors from ST and LT vehicle or nintedanib-treated mice.

(E) Quantification of the percentage of CD31-positive blood vessels that were in contact with NG2-positive perivascular cells in Py2T tumors from ST and LT vehicle or nintedanib-treated mice.

(F) The functionality of blood vessels was assessed by i.v. injection of fluorescein isothiocyanate (FITC)-lectin into Py2T tumor-bearing mice following ST or LT vehicle or nintedanib-treatment. Patent, perfused blood vessels were identified by immunofluorescence staining for CD31 and detection of FITC-lectin and quantified by counting CD31 and lectin double-positive blood vessels.

(G) Hypoxic areas were identified and quantified by immunofluorescence staining for pimonidazole adducts in Py2T tumors from ST and LT vehicle or nintedanib-treated mice.

(H) Representative pictures of the immunofluorescence co-staining for pimonidazole adducts (red) and CD31 (green) on histological sections of tumors from ST and LT vehicle or nintedanib-treated mice. DAPI staining visualizes cell nuclei. The scale bars represent 100 μm.

n = 6–8 mice per group. Mann-Whitney U test. n. s., non-significant; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001. See also Figure S2.