Figure 1.
Control of prostate tumor growth by fractionated irradiation.
(A) Scheme of the conventional fractionated radiation therapy (CFRT) protocol used for treatment of established prostate tumors. (B) Weight of dissected tumors after the indicated time of CFRT. Values = average of n≥6 ± sem. Statistical comparison vs. t14 irradiated. (C) Example of a tumor treated for one week, showing TUNEL+ tumor cells ("T") and normal adjacent tissues ("N") mainly TUNEL-. Tumor and normal tissues were identified with DAPI based on nuclei morphology, size, staining intensity and spatial organization of cells. (D) Pseudo-confocal images of tumors during CFRT, stained for Ki-67/TUNEL/CD31. (E,F) Image quantification of cell proliferation (Ki-67 index, E) and cell death (TUNEL index, F). Statistical comparisons vs. t0.
Figure 2.
Fractionated irradiation reduces hypoxia and increases tumor perfusion.
(A) Pseudo-confocal images of tumors during CFRT, stained for hypoxia (EF5) and endothelial cells (CD31). (B) Image quantification of EF5+ surface in tumors during CFRT. Values represent the average of n≥13 per point ± sem. (C) Pseudo-confocal images of tumors perfused with Hoechst 33342 and 10 kDa/2 MDa dextrans before (t0) or after 2 weeks of CFRT (t14). SYBR green was used as a counterstain of total cell nuclei. (D,E,F) Image quantification of Hoechst+ (D), and medium (E) and large (F) dextran+ surfaces in tumors during CFRT (n = 6). (B,D,E,F). Statistical comparisons vs. t0.
Figure 3.
Maintenance of vascular density and distribution during fractionated irradiation.
(A) Microvessel density in tumors during CFRT. Values represent the average of n≥13 per point ± sem. (B) Distance profile between cells and the closest blood vessel, from tumors during CFRT. Profiles are based on n≥13. Statistical comparisons vs. t0. (C) Pseudo-confocal images of tumor-associated blood vessels (CD31+) stained for TUNEL during CFRT. Arrows: TUNEL+/CD31+ cells. (D) Image quantification of CD31+/TUNEL+ surface. Values represent the average of n≥13 per point ± sem. (E) Representative Z-stack images of 100 µm-thick tumor sections before (t0) or after 2 weeks of CFRT (t14) and stained for blood vessels (CD31+/Fli-1+). (F) Image analysis of blood vessel network from 100 µm-thick tumor sections. Values represent the average of n = 9 per point ± sem.
Figure 4.
Fractionated irradiation induces vascular remodeling.
(A) Pseudo-confocal images of tumor blood vessels during CFRT and stained for ZO-1/CD31 (top) or SMA/CD31 (bottom). (B,C). Image quantification of ZO-1+/CD31+ (B) and peri-CD31+ SMA surfaces (C). Values represent the average of n≥13 per point ± sem. (D) Image quantification of peri-CD31+ desmin surface and frequency of desmin+/SMA+ vessels. (B,E,D) Statistical comparisons vs. t0. (E) Representative confocal images of a blood vessel from a 14-day treated tumor stained for CD31/desmin/SMA. (F) histogram analysis of CD31/desmin/SMA pseudocolor profile of confocal image cross-section.